技术领域Technical field
本发明属于病毒疫苗技术领域，具体涉及一种牛病毒性腹泻病毒的mRNA疫苗及其应用。The invention belongs to the technical field of viral vaccines, and specifically relates to an mRNA vaccine for bovine viral diarrhea virus and its application.
背景技术Background technique
牛病毒性腹泻病毒（Bovine Viral Diarrhea Virus, BVDV）可感染牛引起病毒性腹泻——急性、热性、接触性传染病。BVDV呈世界性分布，感染情况复杂，隐性感染率极高。据估计，其导致的生产损失达687.8美元/头，给全球养牛业造成了巨大损失。BVDV可引起牛生长缓慢、繁殖紊乱、产能降低等多种症状。妊娠50~150天母畜感染后病毒可通过胎盘垂直传播至胎儿，此时由于胎儿免疫系统尚未发育完全，未能识别BVDV而引起免疫耐受，出生后即成为BVDV持续感染牛，且终生带毒，并通过鼻涕、唾液、精液等不断向外界排毒，成为畜群重要的传染源。此病给全球养殖业造成经济损失与生物安全问题，危害巨大。Bovine Viral Diarrhea Virus (BVDV) can infect cattle and cause viral diarrhea - an acute, febrile, contagious disease. BVDV is distributed worldwide, the infection situation is complex, and the latent infection rate is extremely high. It is estimated that the production loss caused by it is US$687.8 per head, causing huge losses to the global cattle industry. BVDV can cause various symptoms such as slow growth, reproductive disorders, and reduced productivity in cattle. The virus can be transmitted vertically to the fetus through the placenta after infection in the mother at 50 to 150 days of gestation. At this time, the fetal immune system is not fully developed and cannot recognize BVDV, causing immune tolerance. After birth, the cow becomes persistently infected with BVDV and carries the virus for life. , and continuously detoxify to the outside world through nasal mucus, saliva, semen, etc., becoming an important source of infection in the livestock herd. This disease has caused economic losses and biosecurity problems to the global breeding industry, causing great harm.
目前使用的国外使用的BVDV疫苗主要包括改良性减毒活疫苗、灭活疫苗、E2亚单位疫苗。在美国，BVDV疫苗主要与牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒等组成多联传统疫苗进行应用。国内商品化BVDV疫苗仅为灭活疫苗，种类单一；可分为BVDV单联灭活疫苗与BVDV、牛传染性鼻气管炎二联灭活疫苗。The BVDV vaccines currently used abroad mainly include modified live attenuated vaccines, inactivated vaccines, and E2 subunit vaccines. In the United States, BVDV vaccines are mainly used in combination with bovine infectious rhinotracheitis virus, bovine respiratory syncytial virus, etc. to form multiple traditional vaccines. Domestic commercial BVDV vaccines are only inactivated vaccines with a single type; they can be divided into BVDV single inactivated vaccine and BVDV and bovine infectious rhinotracheitis double inactivated vaccine.
亚单位疫苗与灭活疫苗存在共有缺点，即需要佐剂，并且需多次免疫，在单剂至抗体水平达到峰值间往往存在免疫空白易感期，且亚单位疫苗存在无法保持天然免疫原构象等缺点。Subunit vaccines and inactivated vaccines share the same shortcomings, that is, they require adjuvants and multiple immunizations. There is often an immune blank susceptibility period between a single dose and the peak antibody level, and subunit vaccines cannot maintain the conformation of the natural immunogen. and other shortcomings.
发明内容Contents of the invention
本发明的目的是为了提高牛病毒性腹泻病毒mRNA疫苗的免疫保护作用。The purpose of the present invention is to improve the immune protective effect of bovine viral diarrhea virus mRNA vaccine.
本发明提供一种牛病毒性腹泻病毒（Bovine viral diarrhea Virus）的mRNA疫苗，所述mRNA疫苗编码牛病毒性腹泻病毒的E2蛋白或牛病毒性腹泻病毒截短E2蛋白。The present invention provides an mRNA vaccine for Bovine viral diarrhea virus (Bovine viral diarrhea Virus). The mRNA vaccine encodes the E2 protein of Bovine viral diarrhea virus or the truncated E2 protein of Bovine viral diarrhea virus.
进一步地限定，牛病毒性腹泻病毒的E2蛋白的序列如SEQ ID NO.9所示；牛病毒性腹泻病毒截短E2蛋白的序列如SEQ ID NO.10所示。To further limit, the sequence of the E2 protein of bovine viral diarrhea virus is shown in SEQ ID NO. 9; the sequence of the truncated E2 protein of bovine viral diarrhea virus is shown in SEQ ID NO. 10.
进一步地限定，mRNA疫苗由以下成分组成：5’端非翻译区、信号肽序列、牛病毒性腹泻病毒的E2蛋白的编码RNA基因或牛病毒性腹泻病毒截短E2蛋白的的编码RNA基因、3’端非翻译区和多聚腺苷酸。It is further defined that the mRNA vaccine consists of the following components: a 5' untranslated region, a signal peptide sequence, an RNA gene encoding the E2 protein of bovine viral diarrhea virus or an RNA gene encoding a truncated E2 protein of bovine viral diarrhea virus, 3' untranslated region and polyadenylation.
进一步地限定，5’端非翻译区的核苷酸序列如SEQ ID NO.1所示；3’端非翻译区的核苷酸序列如SEQ ID NO.2所示；多聚腺苷酸的核苷酸序列如SEQ ID NO.3所示；信号肽的核苷酸序列如SEQ ID NO.5所示；牛病毒性腹泻病毒的E2蛋白的编码RNA基因序列如SEQ IDNO.15所示；牛病毒性腹泻病毒截短E2蛋白的编码RNA基因序列如SEQ ID NO.16所示。To further limit, the nucleotide sequence of the 5'-end untranslated region is shown in SEQ ID NO.1; the nucleotide sequence of the 3'-end untranslated region is shown in SEQ ID NO.2; the polyadenylated The nucleotide sequence is shown in SEQ ID NO.3; the nucleotide sequence of the signal peptide is shown in SEQ ID NO.5; the RNA gene sequence encoding the E2 protein of bovine viral diarrhea virus is shown in SEQ ID NO.15; The sequence of the RNA gene encoding the truncated E2 protein of bovine viral diarrhea virus is shown in SEQ ID NO. 16.
本发明提供牛病毒性腹泻病毒的E2蛋白的序列或牛病毒性腹泻病毒截短E2蛋白的序列在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用，牛病毒性腹泻病毒的E2蛋白的氨基酸序列如SEQ ID NO.9所示；牛病毒性腹泻病毒截短E2蛋白的氨基酸序列如SEQ ID NO.10所示。The present invention provides the application of the sequence of the E2 protein of the bovine viral diarrhea virus or the sequence of the truncated E2 protein of the bovine viral diarrhea virus in the preparation of an mRNA vaccine for preventing or treating bovine respiratory disease syndrome. The E2 protein of the bovine viral diarrhea virus The amino acid sequence of is shown in SEQ ID NO.9; the amino acid sequence of the truncated E2 protein of bovine viral diarrhea virus is shown in SEQ ID NO.10.
本发明提供一种含有编码上述的mRNA疫苗的DNA分子的重组质粒或重组微生物细胞在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用。The present invention provides the application of a recombinant plasmid or recombinant microbial cell containing a DNA molecule encoding the above-mentioned mRNA vaccine in preparing an mRNA vaccine for preventing or treating bovine respiratory disease syndrome.
本发明提供一种含有上述的mRNA疫苗的脂质纳米颗粒在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用。The present invention provides an application of lipid nanoparticles containing the above-mentioned mRNA vaccine in preparing an mRNA vaccine for preventing or treating bovine respiratory disease syndrome.
进一步地限定，引发牛呼吸道疾病综合征的病毒是牛病毒性腹泻病毒。Further defined, the virus causing bovine respiratory disease syndrome is bovine viral diarrhea virus.
本发明提供一种上述的mRNA疫苗在制备牛病毒性腹泻病毒的抗体中的应用。The present invention provides an application of the above-mentioned mRNA vaccine in preparing antibodies to bovine viral diarrhea virus.
有益效果：Beneficial effects:
1.疫苗设计上1. Vaccine design
本研究以中国BVDV流行亚型1a基因亚型毒株E2蛋白基因序列为基础，通过密码子优化以提高翻译效率。在编码E2蛋白的RNA序列前加上信号肽以提高蛋白分泌量，并且在信号肽序列前端添加T7启动子、5’UTR、“cap 1”结构，在编码E2蛋白的RNA序列后端增加3’UTR和多聚腺苷酸尾（poly A）等元件以提高mRNA疫苗翻译效率和稳定性，构建表达E2靶抗原候选mRNA疫苗。并且通过表达截短后的E2蛋白构建高分泌水平的候选mRNA疫苗。This study is based on the E2 protein gene sequence of the Chinese BVDV epidemic subtype 1a gene subtype strain, and improves translation efficiency through codon optimization. A signal peptide is added before the RNA sequence encoding the E2 protein to increase protein secretion, and the T7 promoter, 5'UTR, and "cap 1" structure are added to the front end of the signal peptide sequence, and 3 is added to the back end of the RNA sequence encoding the E2 protein. 'UTR and polyadenylate tail (poly A) and other elements to improve the translation efficiency and stability of the mRNA vaccine, and construct a candidate mRNA vaccine expressing the E2 target antigen. And a candidate mRNA vaccine with high secretion levels was constructed by expressing the truncated E2 protein.
通过体外细胞转染实验与Western Blot实验验证了构建的候选mRNA疫苗在HEK-293T细胞内的表达以及胞外分泌表达，并验证了E2蛋白的二聚体形式， 表达截短E2蛋白的BVDV tE2候选mRNA疫苗表达更多的分泌E2单体蛋白与E2二聚体蛋白。The expression of the constructed candidate mRNA vaccine in HEK-293T cells and the extracellular secretion expression were verified through in vitro cell transfection experiments and Western Blot experiments, and the dimer form of the E2 protein was verified. The BVDV tE2 candidate expressing the truncated E2 protein was verified The mRNA vaccine expresses more secreted E2 monomer protein and E2 dimer protein.
2.免疫效果2.Immune effect
将验证表达的质粒通过体外转录制备mRNA。通过脂质纳米颗粒包装、电镜检测等质量分析，制备成BVDV E2 mRNA疫苗。将构建好的候选mRNA免疫小鼠和犊牛，均可诱导产生针对BVDV的高水平IgG抗体和中和抗体，并且其对于BVDV-1多种不同基因亚型毒株具有交叉保护性。安全性实验表明制备的mRNA候选疫苗具备安全性。牛的攻毒实验表明，BVDV E2mRNA疫苗对国内流行基因亚型BVDV-1b JL毒株具有良好的保护作用，表明了该mRNA疫苗对异源现地流行毒株具有交叉保护作用。The expression-verified plasmid was used to prepare mRNA through in vitro transcription. The BVDV E2 mRNA vaccine was prepared through quality analysis such as lipid nanoparticle packaging and electron microscopy detection. Immunizing mice and calves with the constructed candidate mRNA can induce the production of high-level IgG antibodies and neutralizing antibodies against BVDV, and it has cross-protection against multiple different genetic subtypes of BVDV-1 strains. Safety experiments show that the prepared mRNA vaccine candidate is safe. The cattle challenge experiment showed that the BVDV E2 mRNA vaccine has a good protective effect against the domestically prevalent genetic subtype BVDV-1b JL strain, indicating that the mRNA vaccine has a cross-protective effect against heterologous local circulating strains.
附图说明Description of the drawings
图1抗原表达载体质粒图谱；Figure 1 Antigen expression vector plasmid map;
图2是抗原表达载体质粒表达验证实验中的Western Blot检测结果；Figure 2 is the Western Blot detection result in the plasmid expression verification experiment of the antigen expression vector;
图3是体外转录mRNA琼脂糖凝胶电泳结果图；Figure 3 is a picture of the results of in vitro transcribed mRNA agarose gel electrophoresis;
图4是体外转录 mRNA蛋白表达验证实验中的Western Blot检测结果；Figure 4 is the Western Blot detection result in the in vitro transcription mRNA protein expression verification experiment;
图5是LNP-mRNA包装后的电镜分析结果图；图5（A）是牛病毒性腹泻病毒的E2蛋白的LNP-mRNA包装后，图5（B）是牛病毒性腹泻病毒截短E2蛋白的LNP-mRNA包装后；Figure 5 is the electron microscope analysis result after LNP-mRNA packaging; Figure 5 (A) is the LNP-mRNA of the E2 protein of bovine viral diarrhea virus after packaging, Figure 5 (B) is the truncated E2 protein of bovine viral diarrhea virus After packaging of LNP-mRNA;
图6是BVDV mRNA疫苗与灭活疫苗免疫小鼠的E2特异性IgG抗体-时间曲线图；Figure 6 is a E2-specific IgG antibody-time curve graph of mice immunized with BVDV mRNA vaccine and inactivated vaccine;
图7是BVDV mRNA疫苗与灭活疫苗免疫小鼠的NADL特异性中和抗体抗体-时间曲线图；Figure 7 is an antibody-time curve of NADL-specific neutralizing antibodies in mice immunized with BVDV mRNA vaccine and inactivated vaccine;
图8是BVDV mRNA疫苗与灭活疫苗免疫小鼠2免后两周的E2特异性IgG抗体、NADL特异性中和抗体结果；Figure 8 shows the results of E2-specific IgG antibodies and NADL-specific neutralizing antibodies two weeks after immunization of mice immunized with BVDV mRNA vaccine and inactivated vaccine;
图9是BVDV mRNA疫苗免疫鼠血清的交叉中和实验结果；Figure 9 is the cross-neutralization experimental results of the serum of mice immunized with BVDV mRNA vaccine;
图10是 BVDV mRNA疫苗免疫牛的NADL特异性中和抗体结果；Figure 10 shows the results of NADL-specific neutralizing antibodies in cattle immunized with BVDV mRNA vaccine;
图11是BVDV mRNA疫苗与BVDV灭活疫苗免疫牛的NADL特异性中和抗体结果。Figure 11 shows the results of NADL-specific neutralizing antibodies in cattle immunized with BVDV mRNA vaccine and BVDV inactivated vaccine.
具体实施方式Detailed ways
实施例1. 牛病毒性腹泻病毒mRNA疫苗的抗原表达载体的构建Example 1. Construction of antigen expression vector for bovine viral diarrhea virus mRNA vaccine
一、抗原表达载体质粒的构建方法如下：1. The construction method of antigen expression vector plasmid is as follows:
（1）交由金斯瑞生物公司合成以下基因序列：从5’至3’端分别为T7启动子、5’UTR（SEQ ID NO .1）、kozak序列、tPA信号肽序列（SEQ ID NO .4）、E2序列（SEQ ID NO .6），构建至pCAGGS载体上，合成质粒命名为pCAGGS-1a-E2；(1) The following gene sequences were synthesized by GenScript Biologics: from the 5' to the 3' end are the T7 promoter, 5'UTR (SEQ ID NO.1), kozak sequence, and tPA signal peptide sequence (SEQ ID NO. .4), E2 sequence (SEQ ID NO.6), constructed into the pCAGGS vector, and the synthetic plasmid was named pCAGGS-1a-E2;
（2）使用New England Biolabs公司XhoⅠ和KpnⅠ限制性内切酶进行双酶切，通过琼脂糖凝胶电泳回收包含E2基因的DNA片段与pCAGGS载体片段。(2) Use New England Biolabs'Xho Ⅰ andKpn Ⅰ restriction endonucleases for double digestion, and recover the DNA fragment containing the E2 gene and the pCAGGS vector fragment through agarose gel electrophoresis.
（3）在哈尔滨睿博兴科公司合成条引物（F: ATATAAGAGCCACCGCTAGCCTCGAGGCCACCATGGACGCCATGAA；R-tE2: GAGGCTCCAGCCTATTATCAGATATCGGTACCTCAAAAGTAGTCCCGGTGG；）(3) Primers were synthesized in Harbin Ruibo Xingke Company (F: ATATAAGAGCCACCGCTAGCCTCGAGGCCACCATGGACGCCATGAA; R-tE2: GAGGCTCCAGCCTATTATCAGATATCGGTACCTCAAAAGTAGTCCCGGTGG;)
利用TOYOBO公司的KOD酶以回收包含E2基因的DNA片段为模板进行PCR扩增反应得到截短E2蛋白（tE2）基因序列（SEQ ID NO .7）。The TOYOBO company's KOD enzyme was used to perform PCR amplification reaction using the recovered DNA fragment containing the E2 gene as a template to obtain the truncated E2 protein (tE2) gene sequence (SEQ ID NO.7).
在哈尔滨睿博兴科公司合成引物（F: ATATAAGAGCCACCGCTAGCCTCGAGGCCACCATGGACGCCATGAA；R-EGFP：GAGGCTCCAGCCTATTATCAGATATCGGTACCCTACTTGTACAGCTCGTC），以实验室保存的EGFP质粒为模板通过TOYOBO公司的KOD酶进行PCR扩增反应得到EGFP基因序列（SEQ ID NO .5）。The primers (F: ATATAAGAGCCACCGCTAGCCTCGAGGCCACCATGGACGCCATGAA; R-EGFP: GAGGCTCCAGCCTATTATCAGATATCGGTACCCTACTTGTACAGCTCGTC) were synthesized in Harbin Ruibo Xingke Company, and the EGFP plasmid stored in the laboratory was used as a template to perform a PCR amplification reaction using the KOD enzyme of TOYOBO Company to obtain the EGFP gene sequence (SEQ ID NO. 5).
（4）将PCR产物进行1%琼脂糖凝胶电泳，利用Axygen公司凝胶回收试剂盒回收PCR片段DNA。(4) Perform 1% agarose gel electrophoresis on the PCR product, and use the Axygen gel recovery kit to recover the PCR fragment DNA.
（5）通过Takara公司infusion酶进行同源重组，分别将（2）中回收的pCAGGS载体片段与（4）回收的截短tE2 DNA片段与EGFP DNA片段进行同源重组反应，50℃反应15min后取2.5uL产物加入置擎科公司的感受态DH5α感受态细胞中，冰浴30min，42℃热激90s，冰浴2min，加入1mL LB培养液， 37℃摇床 200rpm振摇30min后，2000rpm离心10min后，用100uLLB重悬菌体后涂布与卡纳抗性固体琼脂培养平板上，待第二天挑取菌落。(5) Perform homologous recombination using the infusion enzyme of Takara Company. The pCAGGS vector fragment recovered in (2), the truncated tE2 DNA fragment recovered in (4) and the EGFP DNA fragment are subjected to homologous recombination reaction. After reacting at 50°C for 15 minutes, Take 2.5uL of the product and add it to the competent DH5α competent cells of Chiqingke Company, incubate on ice for 30 minutes, heat shock at 42℃ for 90s, ice bath for 2min, add 1mL of LB culture medium, shake at 37℃ at 200rpm for 30min, and centrifuge at 2000rpm. After 10 minutes, resuspend the bacterial cells with 100uLLB and spread it on the Kana resistant solid agar culture plate. The colonies will be picked the next day.
（6）通过AxyPrep公司的质粒DNA小量试剂盒分别提取2mL菌液中的质粒，质粒分别命名为pCAGGS-1a-tE2（连接有tE2 DNA片段的pCAGGs载体）；pCAGGS-EGFP（连接EGFP的pCAGGs载体）。使用New England Biolabs公司XhoⅠ和KpnⅠ限制性内切酶进行双酶切鉴定，通过哈尔滨睿博兴科公司测序鉴定重组质粒序列正确。重组质粒pCAGGS-EGFP 、pCAGGS-1a-E2、pCAGGS-1a-tE2如图1所示。(6) Extract plasmids from 2 mL of bacterial fluid using AxyPrep's plasmid DNA mini kit. The plasmids are named pCAGGS-1a-tE2 (pCAGGs vector connected to tE2 DNA fragment); pCAGGS-EGFP (pCAGGs connected to EGFP). carrier).Xho Ⅰ andKpn Ⅰ restriction endonucleases from New England Biolabs were used for double enzyme digestion identification, and the correct sequence of the recombinant plasmid was confirmed by sequencing from Harbin Ruibo Xingke Company. The recombinant plasmids pCAGGS-EGFP, pCAGGS-1a-E2, and pCAGGS-1a-tE2 are shown in Figure 1.
实施例2. mRNA转录验证试验Example 2. mRNA transcription verification test
将实施例1中所获得的质粒（pCAGGS-EGFP ；pCAGGS-1a-E2；pCAGGS-1a-tE2）分别用New England Biolabs公司的BsaI限制性核酸内切酶进行酶切，使质粒线性化。下一步进行线性化DNA的纯化，加入 1/10 体积3 M醋酸钠 (pH=5.2)以及3体积无水乙醇，将体系放置于–20°C 1h，通过离心机15000rpm转速4℃条件下离心30min，弃去上清，用DEPC水重悬DNA，测定DNA浓度。The plasmids (pCAGGS-EGFP; pCAGGS-1a-E2; pCAGGS-1a-tE2) obtained in Example 1 were digested withBsa I restriction endonuclease from New England Biolabs to linearize the plasmids. Next, purify the linearized DNA, add 1/10 volume of 3 M sodium acetate (pH=5.2) and 3 volumes of absolute ethanol, place the system at –20°C for 1 hour, and centrifuge at 15,000 rpm at 4°C. After 30 minutes, discard the supernatant, resuspend the DNA in DEPC water, and measure the DNA concentration.
使用Thermofisher公司的mMESSAGE mMACHINE™ T7 Transcription Kit产品进行体外转录。在PCR仪器中37℃ 反应2h。加入试剂盒中的TURBO DNase 1uL，混匀后在PCR仪器中37℃反应15min。使用Qiagen公司的RNA纯化试剂盒进行纯化，使用DEPC水洗脱吸附柱上的RNA，通过Nanodrop仪器测RNA浓度，并将RNA储存于-80℃。Use Thermofisher's mMESSAGE mMACHINE™ T7 Transcription Kit product for in vitro transcription. React in a PCR instrument at 37°C for 2 hours. Add 1uL of TURBO DNase in the kit, mix well, and react in a PCR instrument at 37°C for 15 minutes. Use Qiagen's RNA purification kit for purification, use DEPC water to elute the RNA on the adsorption column, measure the RNA concentration with a Nanodrop instrument, and store the RNA at -80°C.
配制1%琼脂糖凝胶，通过琼脂糖凝胶电泳验证体外转录的RNA质量。结果如图3所示，体外转录的EGFP mRNA、1a-E2 mRNA与1a-tE2 mRNA条带清晰单一。体外转录的EGFPmRNA序列如SEQ ID NO.20所示，1a-E2 mRNA序列如SEQ ID NO.18所示，1a-tE2 mRNA序列如SEQ ID NO.19所示。Prepare a 1% agarose gel and verify the quality of the in vitro transcribed RNA through agarose gel electrophoresis. The results are shown in Figure 3. The in vitro transcribed EGFP mRNA, 1a-E2 mRNA and 1a-tE2 mRNA bands are clear and single. The in vitro transcribed EGFPmRNA sequence is shown in SEQ ID NO.20, the 1a-E2 mRNA sequence is shown in SEQ ID NO.18, and the 1a-tE2 mRNA sequence is shown in SEQ ID NO.19.
5’端非翻译区（5’UTR）的DNA序列如SEQ ID NO.1所示：GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG。The DNA sequence of the 5' untranslated region (5'UTR) is shown in SEQ ID NO.1: GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG.
3’端非翻译区（3’UTR）的DNA序列如SEQ ID NO.2所示GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG。The DNA sequence of the 3' untranslated region (3'UTR) is shown in SEQ ID NO. 2: GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG.
多聚腺苷酸（poly A）其序列如SEQ ID NO.3所示，含有104个碱基A：AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。Polyganide (Poly A) its sequence is shown in SEQ ID No.3, containing 104 bases AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAActctEctctctctthctableablectctablectablectablectctctctctctctctctableableaablea
T7启动子的DNA序列：TAATACGACTCACTATAGG；DNA sequence of T7 promoter: TAATACGACTCACTATAGG;
信号肽tPA序列的DNA序列如SEQ ID NO.4所示：ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC。The DNA sequence of the signal peptide tPA sequence is shown in SEQ ID NO.4: ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC.
EGFP核苷酸编码基因序列如SEQ ID NO.5所示：ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAG。The EGFP nucleotide encoding gene sequence is shown in SEQ ID NO.5: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCG ACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACT TCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAG.
密码子优化后的E2抗原DNA序列如SEQ ID NO.6所示：ATGCACCTGGACTGTAAGCCTGAGTTCAGCTACGCCATCGCCAAAGACGAGCGGATCGGCCAGCTGGGAGCTGAAGGCCTCACCACCACATGGAAGGAATACAGCCCCGGAATGAAACTGGAAGATACCATGGTCATCGCCTGGTGCGAGGATGGCAAGCTTATGTACCTGCAGCGGTGCACCAGAGAAACAAGATACCTGGCCATCCTGCACACCCGGGCTCTGCCTACCAGCGTGGTTTTCAAGAAGCTGTTCGATGGAAGAAAGCAGGAGGACGTGGTGGAAATGAACGACAATTTTGAGTTCGGCCTGTGCCCTTGTGACGCCAAGCCAATAGTGCGGGGCAAGTTCAACACCACGCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCTATCGGCTGGACCGGCACCGTGTCTTGCACCAGCTTCAACATGGATACACTGGCAACCACAGTGGTTAGAACCTACCGGAGAAGCAAGCCCTTCCCTCACAGACAGGGCTGCATCACACAGAAGAACCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGCAATTGGACCTGTGTGCCTGGCGACCAGCTGCTGTATAAGGGCGGCAGCATCGAGAGCTGCAAGTGGTGCGGCTACCAATTTAAAGAAAGCGAAGGCCTGCCCCACTACCCCATTGGAAAATGCAAGCTGGAAAACGAGACAGGCTACAGACTGGTGGACAGCACCAGCTGTAATAGAGAAGGTGTGGCCATCGTGCCACAGGGCACCCTGAAGTGCAAGATCGGCAAAACAACAGTGCAGGTGATCGCCATGGACACAAAGCTGGGCCCAATGCCTTGCAGACCCTACGAGATCATCAGCAGCGAGGGCCCTGTGGAAAAGACAGCCTGCACTTTTAACTACACCAAAACCCTGAAGAATAAGTACTTCGAGCCTAGAGACTCCTACTTCCAGCAGTACATGCTGAAGGGCGAGTATCAGTACTGGTTTGACCTGGAAGTGACAGACCACCACCGGGACTACTTTGCCGAGAGCATCCTGGTGGTGGTGGTCGCCCTGCTCGGAGGCAGATACGTGCTGTGGCTGCTCGTGACCTACATGGTGCTCAGCGAGCAGAAAGCCCTGGGC。The codon-optimized E2 antigen DNA sequence is shown in SEQ ID NO.6: ATGCACCTGGACTGTAAGCCTGAGTTCAGCTACGCCATCGCCAAAGACGAGCGGATCGGCCAGCTGGGAGCTGAAGGCCTCACCACCACATGGAAGGAATACAGCCCCGGAATGAAACTGGAAGATACCATGGTCATCGCCTGGTGCGAGGATGGCAAGCTTATGTACCTGCAGCGGTGCACCAGAGAAACAAGATACCTGGCCATCCTGCACACCCGG GCTCTGCCTACCAGCGTGGTTTTCAAGAAGCTGTTCGATGGAAGAAAGCAGGAGGACTGGTGGAAATGAACGACAATTTTGAGTTCGGCCTGTGCCCTTGTGACCAAGCCAATAGTGCGGGGCAAGTTCAACACCACGCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCTATCGGCTGGACCGGCACCGTGTCTTGCACCAGCTTCAACATGGATACACTGGCAACCACAGTGGTTAGAACCTACCGGAGA AGCAAGCCCTTCCCTCACAGACAGGGCTGCATCACACAGAAGAACCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGCAATTGGACCTGTGTGCCTGGCGACCAGCTGCTGTATAAGGGCGGCAGCATCGAGAGCTGCAAGTGGTGCGGCTACCAATTTAAAGAAAGCGAAGGCCTGCCCCACTACCCATTGGAAAATGCAAGCTGGAAAACGAGACAGGCTACAGACTGGTGGACAGCACCAGCTGTAATAGAGAAG GTGTGGCCATCGTGCCACAGGGCACCCTGAAGTGCAAGATCGGCAAAACAACAGTGCAGGTGATCGCCATGGACACAAAGCTGGGCCCAATGCCTTGCAGACCCTACGAGATCATCAGCAGCGAGGGCCCTGTGGAAAAGACAGCCTGCACTTTTAACTACACCAAAACCCTGAAGAATAAGTACTTCGAGCCTAGAGACTCCTACTTCCAGCAGTACATGCTGAAGGGCGAGTATCAGTACTGGTTTGACCTGGAAGTGACAGACC ACCACCGGGACTACTTTGCCGAGAGCATCCTGGTGGTGGTGGTCGCCCTGCTCGGAGGCAGATACGTGCTGTGGCTGCTCGTGACCTACATGGTGCTCAGCGAGCAGAAAGCCCTGGGC.
密码子优化后的截短E2蛋白tE2抗原DNA序列如SEQ ID NO.7所示：ATGCACCTGGACTGTAAGCCTGAGTTCAGCTACGCCATCGCCAAAGACGAGCGGATCGGCCAGCTGGGAGCTGAAGGCCTCACCACCACATGGAAGGAATACAGCCCCGGAATGAAACTGGAAGATACCATGGTCATCGCCTGGTGCGAGGATGGCAAGCTTATGTACCTGCAGCGGTGCACCAGAGAAACAAGATACCTGGCCATCCTGCACACCCGGGCTCTGCCTACCAGCGTGGTTTTCAAGAAGCTGTTCGATGGAAGAAAGCAGGAGGACGTGGTGGAAATGAACGACAATTTTGAGTTCGGCCTGTGCCCTTGTGACGCCAAGCCAATAGTGCGGGGCAAGTTCAACACCACGCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCTATCGGCTGGACCGGCACCGTGTCTTGCACCAGCTTCAACATGGATACACTGGCAACCACAGTGGTTAGAACCTACCGGAGAAGCAAGCCCTTCCCTCACAGACAGGGCTGCATCACACAGAAGAACCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGCAATTGGACCTGTGTGCCTGGCGACCAGCTGCTGTATAAGGGCGGCAGCATCGAGAGCTGCAAGTGGTGCGGCTACCAATTTAAAGAAAGCGAAGGCCTGCCCCACTACCCCATTGGAAAATGCAAGCTGGAAAACGAGACAGGCTACAGACTGGTGGACAGCACCAGCTGTAATAGAGAAGGTGTGGCCATCGTGCCACAGGGCACCCTGAAGTGCAAGATCGGCAAAACAACAGTGCAGGTGATCGCCATGGACACAAAGCTGGGCCCAATGCCTTGCAGACCCTACGAGATCATCAGCAGCGAGGGCCCTGTGGAAAAGACAGCCTGCACTTTTAACTACACCAAAACCCTGAAGAATAAGTACTTCGAGCCTAGAGACTCCTACTTCCAGCAGTACATGCTGAAGGGCGAGTATCAGTACTGGTTTGACCTGGAAGTGACAGACCACCACCGGGACTACTTT。The codon-optimized tE2 antigen DNA sequence of the truncated E2 protein is shown in SEQ ID NO.7: ATGCACCTGGACTGTAAGCCTGAGTTCAGCTACGCCATCGCCAAAGACGAGCGGATCGGCCAGCTGGGAGCTGAAGGCCTCACCACCACATGGAAGGAATACAGCCCCGGAATGAAACTGGAAGATACCATGGTCATCGCCTGGTGCGAGGATGGCAAGCTTATGTACCTGCAGCGGTGCACCAGAGAAACAAGATACCTGGCCAT CCTGCACACCCGGGCTCTGCCTACCAGCGGTGGTTTTCAAGAAGCTGTTCGATGGAAGAAAGCAGGAGGACGTGGTGGAAATGAACGACAATTTTGAGTTCGGCCTGTGCCCTTGTGACGCCAAGCCAATAGTGCGGGGCAAGTTCAACACCACGCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCTATCGGCTGGACCGGCACCGTGTCTTGCACCAGCTTCAACATGGATACACTGGCAACCACAGTGGTTAGA ACCTACCGGAGAAGCAAGCCCTTCCCTCACAGACAGGGCTGCATCACACAGAAGAACCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGCAATTGGACCTGTGTGCCTGGCGACCAGCTGCTGTATAAGGGCGGCAGCATCGAGAGCTGCAAGTGGTGCGGCTACCAATTTAAAGAAAGCGAAGGCCTGCCCCACTACCCATTGGAAAATGCAAGCTGGAAAACGAGACAGGCTACAGACTGGTGGACAGCACCAGCT GTAATAGAGAAGGTTGTGGCCATCGTGCCACAGGGCACCCTGAAGTGCAAGATCGGCAAAACAACAGTGCAGGTGATCGCCATGGACACAAAGCTGGGCCCAATGCCTTGCAGACCCTACGAGATCATCAGCAGCGAGGGCCCTGTGGAAAAGACAGCCTGCACTTTTAACTACACCAAAACCCTGAAGAATAAGTACTTCGAGCCTAGAGACTCCTACTTCCAGCAGTACATGCTGAAGGGCGAGTATCAGTACTGGTTTGACCTG GAAGTGACAGACCACCACCGGGACTACTTT.
密码子优化后的E2抗原氨基酸序列如SEQ ID NO.9所示：The codon-optimized amino acid sequence of the E2 antigen is shown in SEQ ID NO.9:
MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYVLWLLVTYMVLSEQKALG。MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQ FKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYVLWLLVTYMVLSEQKALG.
密码子优化后的截短E2蛋白 tE2抗原氨基酸序列如SEQ ID NO.10所示：The amino acid sequence of the tE2 antigen of the truncated E2 protein after codon optimization is shown in SEQ ID NO.10:
MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYF。MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQ FKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYF.
5’端非翻译区（5’UTR）的RNA序列如SEQ ID NO.11所示The RNA sequence of the 5’ untranslated region (5’UTR) is shown in SEQ ID NO.11
GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAG。GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAG.
3’ 端非翻译区（3’UTR）的RNA序列如SEQ ID NO.12所示：GAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUG。The RNA sequence of the 3’ untranslated region (3’UTR) is shown in SEQ ID NO. 12: GAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUG.
多聚腺苷酸（poly A）的RNA序列如SEQ ID NO.13所示：The RNA sequence of polyadenylate (poly A) is shown in SEQ ID NO.13:
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.
tPA信号肽的RNA序列如SEQ ID NO.14所示：The RNA sequence of tPA signal peptide is shown in SEQ ID NO.14:
AUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCC。AUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCC.
密码子优化后的E2抗原RNA序列如SEQ ID NO.15所示：The codon-optimized E2 antigen RNA sequence is shown in SEQ ID NO.15:
AUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUGCCGAGAGCAUCCUGGUGGUGGUGGUCGCCCUGCUCGGAGGCAGAUACGUGCUGUGGCUGCUCGUGACCUACAUGGUGCUCAGCGAGCAGAAAGCCCUGGGC。AUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCU GUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCACAGACAGG GCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCC ACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACC ACCACCGGGACUACUUUGCCGAGAGCAUCCUGGUGGUGGUGGUCGCCCUGCUCGGAGGCAGAUACGUGCUGUGGCUGCUCGUGACCUACAUGGUGCUCAGCGAGCAGAAAGCCCUGGGC.
截短E2蛋白 tE2抗原RNA序列如SEQ ID NO.16所示：The truncated E2 protein tE2 antigen RNA sequence is shown in SEQ ID NO.16:
AUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUU。AUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCU GUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCACAGACAGG GCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCC ACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACC ACCACCGGGACUACUUU.
EGFP蛋白RNA序列如SEQ ID NO.17所示：The EGFP protein RNA sequence is shown in SEQ ID NO.17:
AUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAG。AUGGUGAGCAAGGGCGAGGAGCUGUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUU CAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACA ACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAG.
mRNA疫苗中编码E2抗原的1a-E2 mRNA分子序列如SEQ ID NO.18所示：The sequence of the 1a-E2 mRNA molecule encoding the E2 antigen in the mRNA vaccine is shown in SEQ ID NO.18:
5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUGCCGAGAGCAUCCUGGUGGUGGUGGUCGCCCUGCUCGGAGGCAGAUACGUGCUGUGGCUGCUCGUGACCUACAUGGUGCUCAGCGAGCAGAAAGCCCUGGGCUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUGCCGAGAGCAUCCUGGUGGUGGUGGUCGCCCUGCUCGGAGGCAGAUACGUGCUGUGGCUGCUCGUGACCUACAUGGUGCUCAGCGAGCAGAAAGCCCUGGGCUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
mRNA疫苗中编码截短E2蛋白的1a-tE2 mRNA分子序列如SEQ ID NO.19所示：The sequence of the 1a-tE2 mRNA molecule encoding the truncated E2 protein in the mRNA vaccine is shown in SEQ ID NO.19:
5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUGUUCGUGAGCGCCUCCAUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGG AAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGC UGCUGAACGGCCCCUCCAGAUGGUGGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGA GAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCC UGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCGGGACUACUUUUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCCCUUCCUGCACC CGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.
体外转录完整EGFP mRNA其RNA序列如SEQ ID NO.20所示：The RNA sequence of the complete EGFP mRNA transcribed in vitro is shown in SEQ ID NO.20:
5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAGGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGC GAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACC GCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCCGCCCGACAACCACUACCUGAGCACCCAGUCCGCCGA GCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGACCGCCGCCGGGAUCACUCGGCAUGGACGAGCUGUACAAGUAGGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.
实施例3. E2蛋白表达验证试验Example 3. E2 protein expression verification test
（1）将纯化后的体外转录RNA通过Mirus公司TransIT™-mRNA Transfection Kits试剂转染HEK-293T细胞进行验证表达。将293T细胞均匀铺于六孔板中，使用Mirus公司TransIT™-mRNA Transfection Kits试剂盒进行RNA的转染，每孔转染2 μg mRNA。转染48h后，收集细胞上清液，使用Millipore公司的10kDa浓缩管进行上清液的浓缩，分别加入含DDT的5×loading buffer与不含DTT的5×loading buffer进行样品处理，通过还原性SDS-PAGE与非还原性SDS-PAGE电泳后使用proteintech公司的抗His标签抗体得到WesternBlot结果；(1) Transfect the purified in vitro-transcribed RNA into HEK-293T cells using Mirus TransIT™-mRNA Transfection Kits reagent to verify expression. 293T cells were evenly spread in a six-well plate, and RNA was transfected using Mirus TransIT™-mRNA Transfection Kits. Each well was transfected with 2 μg of mRNA. 48 hours after transfection, collect the cell supernatant, use Millipore's 10kDa concentrator tube to concentrate the supernatant, add 5× loading buffer containing DDT and 5× loading buffer without DTT for sample processing. After SDS-PAGE and non-reducing SDS-PAGE electrophoresis, proteintech's anti-His tag antibody was used to obtain WesternBlot results;
（2）结果如图4所示，与载体对照组pCAGGs-tPA-EGFP mRNA相比，转染了1a-E2mRNA与1a-tE2 mRNA的试验组中均出现了与大小与预期相符的蛋白条带。并且1a-tE2 mRNA试验组上清中的蛋白含量远高于pCAGGs-tPA-E2 mRNA试验组，表明携带tPA信号肽的tE2a靶抗原序列表达蛋白具有高效分泌特征。(2) The results are shown in Figure 4. Compared with the vector control group pCAGGs-tPA-EGFP mRNA, protein bands with sizes consistent with expectations appeared in the test groups transfected with 1a-E2mRNA and 1a-tE2mRNA. . Moreover, the protein content in the supernatant of the 1a-tE2 mRNA test group was much higher than that of the pCAGGs-tPA-E2 mRNA test group, indicating that the tE2a target antigen sequence-expressed protein carrying the tPA signal peptide has the characteristics of efficient secretion.
实施例4.制备mRNA疫苗Example 4. Preparation of mRNA vaccine
通过BsaI限制性核酸内切酶对实施例1中构建成功得到的质粒pCAGGS-1a-E2；pCAGGS-1a-tE2进行单酶切，使其环状DNA线性化。通过3 M醋酸钠 (pH=5.2)、无水乙醇、0.5M EDTA纯化DNA分子：加入 1/10 体积3 M醋酸钠 (PH=5.2)以及3体积无水乙醇，1/20体积0.5 M EDTA，混匀，放置-20℃，15000rpm离心后得到沉淀的DNA分子。使用蛋白酶K进行下一步纯化，去除杂质蛋白。通过苯酚/氯仿方法提取线性化DNA分子，DNA溶解至无核酸酶水以待体外转录。The plasmids pCAGGS-1a-E2 and pCAGGS-1a-tE2 successfully constructed in Example 1 were digested withBsa I restriction endonuclease to linearize their circular DNA. Purify DNA molecules by 3 M sodium acetate (pH=5.2), absolute ethanol, 0.5M EDTA: add 1/10 volume of 3 M sodium acetate (pH=5.2) and 3 volumes of absolute ethanol, 1/20 volume of 0.5 M EDTA , mix well, place at -20°C, and centrifuge at 15,000 rpm to obtain precipitated DNA molecules. Proteinase K is used for the next step of purification to remove impurity proteins. Linearized DNA molecules were extracted by the phenol/chloroform method, and the DNA was dissolved in nuclease-free water for in vitro transcription.
通过诺唯赞公司的T7 High Yield RNA Transcription Kit(N1 -Me-PseudoUTP)产品对上述线性化DNA分子进行体外转录，该反应可获得大量带N1 -Me-Pseudo UTP修饰的mRNA，N1 -Me-Pseudo UTP 修饰目的是为了降低mRNA疫苗诱导的天然免疫反应。接着对体外转录mRNA进行加帽修饰，按近岸公司的Cap 1 Capping System产品说明书操作。通过酚氯仿法提取含帽结构的体外转录mRNA分子，并对其包装制备为LNP-mRNA疫苗。The above-mentioned linearized DNA molecules are transcribed in vitro using Novozant's T7 High Yield RNA Transcription Kit (N1 -Me-PseudoUTP) product. This reaction can obtain a large amount of N1 -Me-Pseudo UTP modified mRNA, N1 -Me- The purpose of Pseudo UTP modification is to reduce the natural immune response induced by mRNA vaccines. Then, the in vitro transcribed mRNA is capped and modified according to the product instructions of Nearshore Company's Cap 1 Capping System. The in vitro-transcribed mRNA molecule containing the cap structure was extracted by the phenol-chloroform method and packaged to prepare an LNP-mRNA vaccine.
具体实验步骤为：首先是醇相的制备，将脂质以阳离子脂质（SM102）:二硬酯酰磷脂酰胆碱（DSPC） : 胆固醇：DMG-2000（购自赛诺邦格公司）=50:10:38.5:1.5 （质量比）比例溶解在无水乙醇中；其次制备水相，pH4.0的柠檬酸缓冲液（50 mM）作为溶解mRNA的溶质；通过微流控设备以醇相:水相(体积比为1:3)进行mRNA的包装。接着以无RNase的PBS缓冲液进行稀释，使用30 KDa超滤管对其进行浓缩换液。加入等体积蔗糖浓度为20%的PBS溶液，将mRNA浓度调整后为60 μg/ml，蔗糖浓度为10%，最后使用0.22 μm滤膜对其过滤，即为制备好的1a-E2、1a-tE2LNP-mRNA疫苗，分装后置于-20℃储存。The specific experimental steps are: first, prepare the alcohol phase, and combine the lipids with cationic lipid (SM102): distearoylphosphatidylcholine (DSPC): cholesterol: DMG-2000 (purchased from Sanobange Company) = Dissolve it in absolute ethanol at a ratio of 50:10:38.5:1.5 (mass ratio); then prepare an aqueous phase, pH 4.0 citric acid buffer (50 mM) as the solute to dissolve mRNA; use the alcohol phase through a microfluidic device :Aqueous phase (volume ratio 1:3) for packaging of mRNA. Then dilute it with RNase-free PBS buffer, and use a 30 KDa ultrafiltration tube to concentrate and change the medium. Add an equal volume of PBS solution with a sucrose concentration of 20%, adjust the mRNA concentration to 60 μg/ml and the sucrose concentration to 10%, and finally filter it with a 0.22 μm filter membrane, which is the prepared 1a-E2 and 1a- tE2LNP-mRNA vaccine, aliquot and store at -20°C.
所述的质体纳米颗粒包含阳离子脂质、二硬酯酰磷脂酰胆碱（DSPC）、 胆固醇和DMG-2000中的至少一种。The plasmid nanoparticles include at least one of cationic lipids, distearoylphosphatidylcholine (DSPC), cholesterol and DMG-2000.
通过电镜检测结果（图5）验证制备的制备后的1a-E2、1a-tE2 LNP-mRNA疫苗颗粒均匀一致。The results of electron microscopy (Figure 5) verified that the prepared 1a-E2 and 1a-tE2 LNP-mRNA vaccine particles were uniform and consistent.
实施例5.小鼠免疫评价实验Example 5. Mouse immunity evaluation experiment
将实施例4制备好的1a-E2 mRNA疫苗与1a-tE2 mRNA疫苗免疫BALB/c小鼠进行免疫效果的评价。将20只购于辽宁长生生物技术股份有限公司的8周龄SPF BALB/c小鼠随机分成44组，5只/组，分别免疫20ug （100uL ）1a-E2 mRNA疫苗、20ug （100uL）1a-tE2 mRNA疫苗、60uL 华威特公司的BVDV（1a-NM01毒株）灭活疫苗；100uL PBS。免疫3周后，使用和首次免疫相同剂量体积的疫苗进行加强免疫，之后每隔两周进行小鼠血液的采集并分离血清进行抗体检测。The 1a-E2 mRNA vaccine and 1a-tE2 mRNA vaccine prepared in Example 4 were immunized into BALB/c mice to evaluate the immune effect. Twenty 8-week-old SPFBALB/c mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were randomly divided into 44 groups, 5 mice/group, and were immunized with 20ug (100uL) 1a-E2 mRNA vaccine and 20ug (100uL) 1a- tE2 mRNA vaccine, 60uL Warwick's BVDV (1a-NM01 strain) inactivated vaccine; 100uL PBS. Three weeks after immunization, the same dose volume of vaccine as the first immunization was used for booster immunization. After that, the blood of mice was collected every two weeks and the serum was separated for antibody detection.
（1）抗E2 IgG抗体和中和抗体的检测(1) Detection of anti-E2 IgG antibodies and neutralizing antibodies
通过基于E2蛋白为包被抗原的间接ELISA方法分析免疫血清中特异性抗E2抗体效价。将纯化后的E2蛋白（33ng/孔）包被ELISA板，将加强免疫后不同时间点所分离的血清进行倍比稀释，加入对应ELISA板中，依次加入HRP-anti-mouse IgG 二抗，及TMB，显色终止后A450检测OD值，测定抗体效价。结果如图6所示，结果表明mRNA疫苗二免后二周的小鼠血清特异性抗E2抗体效价最高，与BVDV灭活疫苗相比，mRNA疫苗可诱导高水平特异性抗E2 IgG抗体，可以有效地诱导机体产生体液免疫应答（图6，表1）。The specific anti-E2 antibody titer in immune serum was analyzed by an indirect ELISA method based on E2 protein as the coating antigen. Coat the ELISA plate with the purified E2 protein (33ng/well), dilute the serum separated at different time points after the booster immunization, add it to the corresponding ELISA plate, add HRP-anti-mouse IgG secondary antibody in sequence, and TMB, after the color development is terminated, detect the OD value with A450 and determine the antibody titer. The results are shown in Figure 6. The results show that the mouse serum specific anti-E2 antibody titer was the highest two weeks after the second immunization of the mRNA vaccine. Compared with the BVDV inactivated vaccine, the mRNA vaccine can induce high levels of specific anti-E2 IgG antibodies. It can effectively induce the body to produce humoral immune response (Figure 6, Table 1).
通过中和试验分析免疫血清中的中和抗体效价。将等体积报告病毒NADL-mcherry与等体积不同稀释度小鼠血清在37℃中和作用1h后，加入至MDBK细胞中，感染2h后换为含2%马血清的DMEM培养液。3天后通过蔡司公司高通量活细胞分析仪分析NADL-mcherry感染范围及细胞数，计算IC50，中和抗体效价定义为中和50% NADL-mcherry感染时的血清稀释倍数。结果如图7，表2所示，mRNA疫苗二免后二周的小鼠anti-NADL nAb效价最高，与BVDV灭活疫苗相比，mRNA疫苗可诱导高水平特异性anti-NADL nAb。Neutralizing antibody titers in immune sera were analyzed by neutralization assay. Equal volumes of reporter virus NADL-mcherry and equal volumes of mouse serum at different dilutions were neutralized at 37°C for 1 hour and then added to MDBK cells. After 2 hours of infection, they were replaced with DMEM culture medium containing 2% horse serum. Three days later, the NADL-mcherry infection range and cell number were analyzed using a Zeiss high-throughput live cell analyzer, and the IC50 was calculated. The neutralizing antibody titer was defined as the serum dilution factor that neutralizes 50% of the NADL-mcherry infection. The results are shown in Figure 7 and Table 2. The anti-NADL nAb titer of mice two weeks after the second immunization with the mRNA vaccine was the highest. Compared with the BVDV inactivated vaccine, the mRNA vaccine can induce high levels of specific anti-NADL nAb.
进一步比较免疫峰值加强免疫后两周的抗体（图8），相较BVDV灭活疫苗，BVDVmRNA疫苗可诱导显著升高的E2特异性IgG抗体、NADL特异性中和抗体。Further comparing the antibodies two weeks after the immune peak booster vaccination (Figure 8), compared with the BVDV inactivated vaccine, the BVDV mRNA vaccine can induce significantly higher levels of E2-specific IgG antibodies and NADL-specific neutralizing antibodies.
表1 BVDV E2 mRNA疫苗与灭活疫苗免疫接种小鼠诱导的E2特异性IgG抗体滴度Table 1 E2-specific IgG antibody titers induced in mice immunized with BVDV E2 mRNA vaccine and inactivated vaccine
表2BVDV E2 mRNA疫苗与灭活疫苗免疫接种小鼠诱导的NADL特异性中和抗体滴度Table 2 NADL-specific neutralizing antibody titers induced in mice immunized with BVDV E2 mRNA vaccine and inactivated vaccine
（2）交叉保护性的检测(2) Detection of cross-protection
通过其他不同基因亚型BVDV毒株进行中和试验，分析免疫BVDV E2 mRNA疫苗小鼠血清中的交叉中和抗体效价。分别将等体积不同基因亚型BVDV毒株VEDEVAC、3877等与等体积最终稀释度为200的小鼠血清在37℃中和作用1h后，加入至MDBK细胞中，感染2h后换为含2%马血清的DMEM培养液。3天后使用VMRD公司的BVDV多抗血清以及invitrogen的488标记anti-goat IgG抗体通过间接免疫荧光实验观察BVDV感染细胞，通过蔡司公司高通量活细胞分析仪分析BVDV感染范围及细胞数，计算200稀释倍数下的BVDV E2 mRNA疫苗免疫小鼠血清对不同基因亚型BVDV毒株的抑制率。结果如图9所示，mRNA疫苗免疫的小鼠血清中对于BVDV-1多种不同基因亚型毒株具有高达90%的抑制率，对不同基因亚型BVDV毒株具有交叉保护性，其可诱导小鼠产生交叉中和抗体。Neutralization tests were conducted with other BVDV strains of different genotypes to analyze the cross-neutralizing antibody titers in the serum of mice immunized with the BVDV E2 mRNA vaccine. Equal volumes of BVDV strains VEDEVAC, 3877, etc. of different genetic subtypes and equal volumes of mouse serum with a final dilution of 200 were neutralized at 37°C for 1 hour, and then added to MDBK cells. After 2 hours of infection, they were replaced with 2% Horse serum in DMEM. Three days later, use VMRD's BVDV polyantiserum and invitrogen's 488-labeled anti-goat IgG antibody to observe BVDV-infected cells through indirect immunofluorescence experiments, analyze the BVDV infection range and cell number with Zeiss' high-throughput live cell analyzer, and calculate 200 Inhibition rates of BVDV E2 mRNA vaccine immunized mouse sera against different genotype subtypes of BVDV strains at dilution multiples. The results are shown in Figure 9. The serum of mice immunized with the mRNA vaccine has an inhibition rate of up to 90% against multiple different genetic subtypes of BVDV-1 strains, and has cross-protection against different genetic subtypes of BVDV strains. It can Induces mice to produce cross-neutralizing antibodies.
（3）细胞免疫反应的检测(3) Detection of cellular immune response
通过细胞因子染色（ICCS）实验检测抗原特异性T细胞以评估细胞免疫，取疫苗免疫后鼠淋巴细胞接种到培养板中，使用E2蛋白肽库刺激。将细胞与抗 CD28抗体在 37 ℃和 5% CO2条件下共刺激。DMSO用作阴性对照。使用PMA/ionomycin作为阳性对照。随后，使用固定/透化试剂盒进行实验，使用以下细胞因子抗体：抗IFN-γ，抗TNF-α，抗IL-2和抗IL-4与脾细胞作用，最后通过流式细胞术分析细胞因子特异性淋巴细胞数量。Antigen-specific T cells were detected through cytokine staining (ICCS) experiments to evaluate cellular immunity. Mouse lymphocytes after vaccine immunization were inoculated into culture plates and stimulated using the E2 protein peptide library. Cells were costimulated with anti-CD28 antibody at 37 °C and 5%CO2 . DMSO was used as a negative control. PMA/ionomycin was used as a positive control. Subsequently, experiments were performed using a fixation/permeabilization kit using the following cytokine antibodies: anti-IFN-γ, anti-TNF-α, anti-IL-2, and anti-IL-4 with splenocytes, and finally the cells were analyzed by flow cytometry. Factor-specific lymphocyte numbers.
结果在加强疫苗接种后数周，实验结果表明，BVDV E2 mRNA疫苗疫苗可诱导表达IFN-γ、IL-2 和 TNF-a 的抗原特异性、多功能 CD8+T 细胞；相比BVDV灭活疫苗，BVDV E2mRNA疫苗诱导了更高的CD4+和CD8+记忆T细胞。Results Several weeks after booster vaccination, experimental results showed that the BVDV E2 mRNA vaccine could induce antigen-specific, multifunctional CD8+ T cells expressing IFN-γ, IL-2 and TNF-a; compared with the BVDV inactivated vaccine , the BVDV E2mRNA vaccine induced higher CD4+ and CD8+ memory T cells.
实施例6.mRNA疫苗安全性实验Example 6. mRNA vaccine safety experiment
对2月龄犊牛进行免疫接种。共10只犊牛，每组各5头犊牛，一组肌肉接种高剂量mRNA疫苗，600μg/头；另一组注射PBS作为对照。首次接种后3周，相同剂量和途径进行第二次接种。观察犊牛身体健康状况。PBS组、mRNA疫苗组犊牛中所有牛健康状态良好、体温正常，表明疫苗的安全性良好。Vaccination of 2-month-old calves. There were 10 calves in total, 5 calves in each group. One group was intramuscularly inoculated with high-dose mRNA vaccine, 600 μg/head; the other group was injected with PBS as a control. Three weeks after the first vaccination, give the second vaccination with the same dose and route. Observe the health of the calf. All calves in the PBS group and the mRNA vaccine group were in good health and had normal body temperatures, indicating that the vaccine was safe.
实施例7.mRNA疫苗免疫牛实验Example 7. Experiment on immunizing cattle with mRNA vaccine
用3～4月龄健康易感牛8头，每组4只牛。其中一组颈部肌肉注射1a-E2 mRNA疫苗200ug、另一组颈部肌肉注射107TCID50BVDV 灭活病毒疫苗。BVDV 灭活病毒疫苗为实验室制备：BVDV病毒在MDBK细胞中培养，四天后收获细胞并于 -80℃ 冻融 3 次，BVDV通过sigma公司的Binary Ethyleneimine（BEI）灭活，按BVDV灭活病毒与MONTANIDETMISA 15A VG佐剂体积比为85%:15%比例制备BVDV灭活疫苗。21日后进行二免，接种剂量同首免。BVDV 灭活病毒疫苗免疫牛组在上一次免疫后4周分别进行了第三次、第四次免疫，接种剂量同首免。Eight healthy susceptible cows aged 3 to 4 months were used, with 4 cows in each group. One group was injected intramuscularly into the neck with 200ug of 1a-E2 mRNA vaccine, and the other group was intramuscularly injected with 107 TCID50 BVDV inactivated virus vaccine into the neck. BVDV inactivated virus vaccine is prepared in the laboratory: BVDV virus is cultured in MDBK cells. After four days, the cells are harvested and frozen and thawed three times at -80°C. BVDV is inactivated by Binary Ethyleneimine (BEI) of sigma company. According to BVDV inactivated virus Prepare BVDV inactivated vaccine with the volume ratio of MONTANIDETM ISA 15A VG adjuvant at a ratio of 85%:15%. The second dose will be given after the 21st, and the vaccination dose will be the same as the first dose. The cattle group immunized with BVDV inactivated virus vaccine received the third and fourth vaccinations 4 weeks after the last vaccination, and the vaccination dose was the same as the first vaccination.
1a-E2 mRNA免疫组在免疫前、第一次免疫后3周，加强免疫后1周分别进行了血液采集并且分离血清。BVDV 灭活疫苗免疫组在第四次免疫1周后进行血液采集并且分离血清。For the 1a-E2 mRNA immunized group, blood was collected and serum was separated before immunization, 3 weeks after the first immunization, and 1 week after the booster immunization. In the BVDV inactivated vaccine immunization group, blood was collected and serum was separated 1 week after the fourth immunization.
通过中和试验分析免疫牛血清中的中和抗体效价。将等体积报告病毒NADL-mcherry与等体积不同稀释度牛血清在37℃中和作用1h后，加入至MDBK细胞中，感染2h后换为含2%马血清的DMEM培养液。3天后通过蔡司公司高通量活细胞分析仪分析NADL-mcherry感染范围及细胞数，计算IC50，中和抗体效价定义为中和50% NADL-mcherry感染时的血清稀释倍数。结果如图10所示，mRNA疫苗在加强免疫后可诱导高水平anti-NADL nAb效价。与BVDV 灭活疫苗相比，mRNA疫苗可诱导约2倍的更高水平anti-NADL nAb（图11、表3）所示。Neutralizing antibody titers in immunized bovine sera were analyzed by neutralization assay. Equal volumes of reporter virus NADL-mcherry and equal volumes of bovine serum at different dilutions were neutralized at 37°C for 1 hour, and then added to MDBK cells. After 2 hours of infection, they were replaced with DMEM culture medium containing 2% horse serum. Three days later, the NADL-mcherry infection range and cell number were analyzed using a Zeiss high-throughput live cell analyzer, and the IC50 was calculated. The neutralizing antibody titer was defined as the serum dilution factor that neutralizes 50% of the NADL-mcherry infection. The results are shown in Figure 10. The mRNA vaccine can induce high levels of anti-NADL nAb titers after booster immunization. Compared with the BVDV inactivated vaccine, the mRNA vaccine induced approximately 2-fold higher levels of anti-NADL nAb (Figure 11, Table 3).
表3 BVDV E2 mRNA疫苗与BVDV灭活疫苗免疫接种牛诱导的NADL特异性中和抗体滴度Table 3 NADL-specific neutralizing antibody titers induced by cattle immunized with BVDV E2 mRNA vaccine and BVDV inactivated vaccine
实施例8.mRNA疫苗攻毒实验Example 8. mRNA vaccine challenge experiment
用3～4月龄健康易感牛4头，每组2只牛。其中一组颈部肌肉注射1a-E2 mRNA疫苗200ug、另一组作为对照注射PBS。21日后进行二免，接种剂量同首免。二免后21日，每头牛鼻内喷雾接种1b - JL株（病毒含量≥106.5FAID50/ml）6ml。攻毒前观察牛只并无任何咳嗽、发烧等临床症状，并通过PCR检测判定牛只为BVDV阴性，攻毒前测量牛只直肠温度作为牛基础体温数值；攻毒前采取牛的血液并检测其白细胞数量作为牛只的白细胞基础数值。攻毒后每日测量牛只体温并隔日采血，进行血液的白细胞数量测定、并进行BVDV病毒分离。Four healthy susceptible cows aged 3 to 4 months were used, 2 cows in each group. One group was injected with 200ug of 1a-E2 mRNA vaccine intramuscularly in the neck, and the other group was injected with PBS as a control. The second dose will be given after the 21st, and the vaccination dose will be the same as the first dose. On the 21st day after the second vaccination, each cow was sprayed intranasally with 6ml of strain 1b-JL (virus content ≥106.5 FAID50 /ml). Before the virus challenge, the cows were observed to have no clinical symptoms such as cough and fever, and the cows were determined to be BVDV negative through PCR testing. The rectal temperature of the cows was measured before the virus challenge as the basal body temperature of the cattle; the blood of the cows was collected and tested before the virus challenge. The number of white blood cells is used as the basic white blood cell value of the cow. After the challenge, the body temperature of the cattle was measured every day and blood was collected every other day to measure the number of white blood cells in the blood and isolate the BVDV virus.
牛的攻毒实验结果为，注射PBS的对照牛在攻毒后皆出现发烧现象，而免疫1a-E2mRNA疫苗牛只体温正常；白细胞计数结果显示，PBS对照组牛攻毒后白细胞数目下降、1a-E2mRNA接种疫苗组白细胞计数显着高于对照组 。The results of the cattle challenge experiment were that the control cattle injected with PBS all developed fever after the challenge, while the body temperature of the cattle immunized with the 1a-E2mRNA vaccine was normal; the white blood cell count results showed that the number of white blood cells in the PBS control cattle decreased after challenge. -The white blood cell count in the E2mRNA vaccination group was significantly higher than that in the control group.
实验结果表明BVDV E2 mRNA疫苗对国内流行基因亚型BVDV-1b JL毒株具有良好的保护作用，表明了该mRNA疫苗对异源现地流行毒株具有交叉保护作用。The experimental results show that the BVDV E2 mRNA vaccine has a good protective effect against the domestically prevalent genetic subtype BVDV-1b JL strain, indicating that the mRNA vaccine has a cross-protective effect against heterologous local circulating strains.
实施例9.mRNA疫苗制备抗体的方法Example 9. Method for preparing antibodies from mRNA vaccine
将制备好的1a-E2 mRNA疫苗与1a-tE2 mRNA疫苗免疫BALB/c小鼠进行单抗的制备。将10只购于辽宁长生生物技术股份有限公司的8周龄SPF BALB/c小鼠随机分成2组，5只/组，分别免疫20ug1a-E2 mRNA疫苗、20ug 1a-tE2 mRNA疫苗。免疫3周后，使用和首次免疫相同剂量体积的疫苗进行加强免疫，之后进行小鼠血液的采集并测定血清抗体效价。取免疫鼠脾细胞与 SP2/0 细胞融合。通过ELISA 方法、间接免疫荧光等筛选阳性细胞克隆，获得稳定分泌特异性单克隆抗体的杂交瘤细胞株，所制备的单抗可用于建立检测 BVDV 病原及抗体的诊断方法。The prepared 1a-E2 mRNA vaccine and 1a-tE2 mRNA vaccine were immunized into BALB/c mice to prepare monoclonal antibodies. Ten 8-week-old SPFBALB/c mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were randomly divided into 2 groups, 5 mice/group, and were immunized with 20ug1a-E2 mRNA vaccine and 20ug 1a-tE2 mRNA vaccine respectively. Three weeks after immunization, booster immunization was carried out with the same dose volume of vaccine as the first immunization, and then the blood of mice was collected and serum antibody titers were measured. Spleen cells from immunized mice were fused with SP2/0 cells. Positive cell clones are screened through ELISA methods, indirect immunofluorescence, etc., and hybridoma cell lines that stably secrete specific monoclonal antibodies are obtained. The prepared monoclonal antibodies can be used to establish diagnostic methods for detecting BVDV pathogens and antibodies.
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