技术领域technical field
本发明涉及一种流感病毒M1蛋白及其编码基因和应用。The invention relates to an influenza virus M1 protein, its coding gene and application.
背景技术Background technique
流行性感冒病毒，简称流感病毒，是一种造成人、狗、马、猪及禽类等患流行性感冒的RNA病毒，在分类学上，流感病毒属于正黏液病毒科，它会造成急性上呼吸道感染，并借由空气迅速的传播，在世界各地常会有周期性的大流行。Influenza virus, referred to as influenza virus, is an RNA virus that causes influenza in humans, dogs, horses, pigs, and poultry. In taxonomy, influenza virus belongs to the Orthomyxoviridae family, and it can cause acute upper respiratory tract infections. infection, and spreads rapidly through the air, there are often periodic pandemics around the world.
流感病毒分为甲（A）、乙（B）、丙（C）三型，是流行性感冒（流感）的病原体。其中甲型流感病毒抗原性易发生变异，多次引起世界性大流行。Influenza viruses are divided into three types: A (A), B (B), and C (C), which are the pathogens of influenza (flu). Among them, the antigenicity of influenza A virus is easy to mutate, causing worldwide pandemics many times.
禽流感是禽流行性感冒的简称，它是一种由甲型流感病毒的一种亚型（也称禽流感病毒）引起的传染性疾病，被国际兽疫局定为甲类传染病，又称真性鸡瘟或欧洲鸡瘟。按病原体类型的不同，禽流感可分为高致病性、低致病性和非致病性禽流感三大类。非致病性禽流感不会引起明显症状，仅使染病的禽鸟体内产生病毒抗体。低致病性禽流感可使禽类出现轻度呼吸道症状，食量减少，产蛋量下降，出现零星死亡。高致病性禽流感最为严重，发病率和死亡率均高，人感染高致病性禽流感死亡率约是60%，家禽感染的死亡率几乎是100%。Avian influenza is the abbreviation of avian influenza. It is an infectious disease caused by a subtype of influenza A virus (also known as avian influenza virus). True fowl plague or European fowl plague. According to the different types of pathogens, avian influenza can be divided into three categories: highly pathogenic, low pathogenic and non-pathogenic avian influenza. Non-pathogenic avian influenza does not cause obvious symptoms and only produces virus antibodies in infected birds. Low-pathogenic avian influenza can cause mild respiratory symptoms, reduced food intake, decreased egg production, and sporadic deaths in poultry. Highly pathogenic avian influenza is the most serious, with high morbidity and mortality. The mortality rate of human infection is about 60%, and the mortality rate of poultry infection is almost 100%.
发明内容Contents of the invention
本发明的目的是提供一种流感病毒M1蛋白及其编码基因和应用。The purpose of the present invention is to provide a kind of influenza virus M1 protein and its encoding gene and application.
本发明提供了一种人工设计的M1蛋白，为如下（a）或（b）或（c）：The present invention provides an artificially designed M1 protein, which is the following (a) or (b) or (c):
（a）由序列表的序列1所示的氨基酸序列组成的蛋白质；(a) a protein consisting of the amino acid sequence shown in Sequence 1 of the Sequence Listing;
（b）由序列表的序列3所示的氨基酸序列组成的蛋白质；(b) a protein consisting of the amino acid sequence shown in Sequence 3 of the Sequence Listing;
（c）将（a）或（b）经过一个或几个氨基酸残基的取代和/或缺失和/或添加且可以形成病毒样颗粒的由其衍生的蛋白质。(c) A protein derived from (a) or (b) that undergoes substitution and/or deletion and/or addition of one or several amino acid residues and can form virus-like particles.
编码所述M1蛋白的基因也属于本发明的保护范围。The gene encoding the M1 protein also belongs to the protection scope of the present invention.
编码所述M1蛋白的基因优选为如下（1）或（2）或（3）或（4）所述的DNA分子：The gene encoding the M1 protein is preferably the DNA molecule described in (1) or (2) or (3) or (4):
（1）序列表的序列2所示的DNA分子；(1) The DNA molecule shown in Sequence 2 of the Sequence Listing;
（2）序列表的序列4所示的DNA分子；(2) The DNA molecule shown in sequence 4 of the sequence listing;
（3）在严格条件下与（1）或（2）限定的DNA序列杂交且编码可以形成病毒样颗粒的蛋白的DNA分子；(3) A DNA molecule that hybridizes to the DNA sequence defined in (1) or (2) under stringent conditions and encodes a protein that can form virus-like particles;
（4）与（1）或（2）限定的DNA序列至少具有90%以上同源性且可以形成病毒样颗粒的蛋白的DNA分子。(4) A DNA molecule that has at least 90% homology with the DNA sequence defined in (1) or (2) and can form a protein of a virus-like particle.
上述严格条件可为在6×SSC，0.5%SDS的溶液中，在65℃下杂交，然后用2×SSC、0.1%SDS和1×SSC、0.1%SDS各洗膜一次。The above stringent conditions can be hybridized at 65°C in a solution of 6×SSC, 0.5% SDS, and then the membrane is washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.
所述M1蛋白形成的病毒样颗粒也属于本发明的保护范围。The virus-like particles formed by the M1 protein also belong to the protection scope of the present invention.
本发明还保护所述M1蛋白或所述M1蛋白形成的病毒样颗粒的应用，为如下（Ⅰ）或（Ⅱ）或（Ⅲ）或（Ⅳ）：（Ⅰ）作为功能蛋白片段的载体；（Ⅱ）制备功能蛋白片段的载体；（Ⅲ）作为细胞骨架；（Ⅳ）制备细胞骨架。所述功能蛋白片段可为具有疫苗功能的蛋白片段，具体可为具有禽流感疫苗功能的蛋白片段，更具体可为HA蛋白片段。所述HA蛋白片段具体可由序列表的序列5自N末端第253至267位氨基酸残基组成。The present invention also protects the application of the M1 protein or the virus-like particles formed by the M1 protein as follows (I) or (II) or (III) or (IV): (I) as a carrier of functional protein fragments; ( Ⅱ) Carrier for preparing functional protein fragments; (Ⅲ) as cytoskeleton; (Ⅳ) preparing cytoskeleton. The functional protein fragment may be a protein fragment with a vaccine function, specifically a protein fragment with an avian influenza vaccine function, more specifically a HA protein fragment. The HA protein fragment may specifically consist of amino acid residues 253 to 267 from the N-terminal of Sequence 5 in the Sequence Listing.
本发明还保护具有所述M1蛋白和功能蛋白片段的融合蛋白。所述功能蛋白片段可为具有疫苗功能的蛋白片段，具体可为具有禽流感疫苗功能的蛋白片段，更具体可为HA蛋白片段。所述HA蛋白片段具体可由序列表的序列5自N末端第253至267位氨基酸残基组成。The invention also protects the fusion protein with the M1 protein and the functional protein fragment. The functional protein fragment may be a protein fragment with a vaccine function, specifically a protein fragment with an avian influenza vaccine function, more specifically a HA protein fragment. The HA protein fragment may specifically consist of amino acid residues 253 to 267 from the N-terminal of Sequence 5 in the Sequence Listing.
所述融合蛋白具体可为如下（d）或（e）或（f）：The fusion protein may specifically be as follows (d) or (e) or (f):
（d）由序列表的序列5自N端第1至267位氨基酸残基组成的蛋白质；(d) A protein consisting of amino acid residues 1 to 267 from the N-terminus of Sequence 5 in the sequence listing;
（e）由序列表的序列5所示的氨基酸序列组成的蛋白质；(e) a protein consisting of the amino acid sequence shown in Sequence 5 of the Sequence Listing;
（f）将（d）或（e）经过一个或几个氨基酸残基的取代和/或缺失和/或添加且可以形成病毒样颗粒的由其衍生的蛋白质。(f) A protein derived from (d) or (e) that undergoes substitution and/or deletion and/or addition of one or several amino acid residues and can form virus-like particles.
编码所述融合蛋白的基因也属于本发明的保护范围。The gene encoding the fusion protein also belongs to the protection scope of the present invention.
编码所述融合蛋白的基因优选为如下（5）或（6）或（7）或（8）所述的DNA分子：The gene encoding the fusion protein is preferably the DNA molecule described in (5) or (6) or (7) or (8):
（5）序列表的序列6自5’末端第1至801位核苷酸所示的DNA分子；(5) The DNA molecule shown in the 1st to 801st nucleotides from the 5' end of Sequence 6 in the sequence listing;
（6）序列表的序列6所示的DNA分子；(6) The DNA molecule shown in sequence 6 of the sequence listing;
（7）在严格条件下与（5）或（6）限定的DNA序列杂交且编码可以形成病毒样颗粒的蛋白的DNA分子；(7) A DNA molecule that hybridizes to the DNA sequence defined in (5) or (6) under stringent conditions and encodes a protein that can form virus-like particles;
（8）与（5）或（6）限定的DNA序列至少具有90%以上同源性且可以形成病毒样颗粒的蛋白的DNA分子。(8) A DNA molecule that has at least 90% homology with the DNA sequence defined in (5) or (6) and can form a protein of a virus-like particle.
所述融合蛋白形成的病毒样颗粒也属于本发明的保护范围。The virus-like particles formed by the fusion protein also belong to the protection scope of the present invention.
本发明还保护所述融合蛋白或所述融合蛋白形成的病毒样颗粒在制备疫苗中的应用。所述疫苗可为禽流感疫苗，具体可为针对禽H9N2亚型流感病毒的禽流感疫苗，进一步具体可为针对AIV-SD株的禽流感疫苗。所述疫苗可为预防和/或治疗疫苗。The present invention also protects the application of the fusion protein or the virus-like particles formed by the fusion protein in the preparation of vaccines. The vaccine may be an avian influenza vaccine, specifically an avian influenza vaccine against the avian H9N2 subtype influenza virus, and more specifically an avian influenza vaccine against the AIV-SD strain. The vaccines may be prophylactic and/or therapeutic vaccines.
本发明还保护一种疫苗，其活性成分为所述融合蛋白或所述融合蛋白形成的病毒样颗粒。所述疫苗可为禽流感疫苗，具体可为针对禽H9N2亚型流感病毒的禽流感疫苗，进一步具体可为针对AIV-SD株的禽流感疫苗。所述疫苗可为预防和/或治疗疫苗。The invention also protects a vaccine whose active ingredient is the fusion protein or the virus-like particle formed by the fusion protein. The vaccine can be an avian influenza vaccine, specifically an avian influenza vaccine against the avian H9N2 subtype influenza virus, and more specifically an avian influenza vaccine against the AIV-SD strain. The vaccines may be prophylactic and/or therapeutic vaccines.
禽H9N2亚型流感病毒（AvianH9N2InfluenzaAVirus）AIV-SD株，已于2011年3月22日保藏于中国微生物菌种保藏管理委员会普通微生物中心（简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号），保藏号为CGMCCNo.4689。禽H9N2亚型流感病毒又称H9N2亚型禽流感病毒。禽H9N2亚型流感病毒（AvianH9N2InfluenzaAVirus）AIV-SD株CGMCCNo.4689简称AIV-SD株。The Avian H9N2 Influenza AVirus AIV-SD strain was deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms (CGMCC for short) on March 22, 2011. The address is: No. 1 Beichen West Road, Chaoyang District, Beijing Court No. 3), the preservation number is CGMCCNo.4689. Avian H9N2 subtype influenza virus is also called H9N2 subtype avian influenza virus. Avian H9N2 subtype influenza virus (AvianH9N2InfluenzaAVirus) AIV-SD strain CGMCCNo.4689 is referred to as AIV-SD strain.
本发明提供的M1蛋白可以作为形成病毒样颗粒的功能组件，将其与其他功能蛋白片段融合，可以形成病毒样颗粒状的融合蛋白，大大增加功能片段的效果。基于该机理，本发明将M1蛋白与HA蛋白片段融合，该融合蛋白形成病毒样颗粒，其对禽流感病毒的保护效果大大优于HA蛋白片段本身。本发明对于疫苗的研制具有重大价值。本发明对于禽流感病毒的预防或治疗具有重大价值。The M1 protein provided by the present invention can be used as a functional component for forming virus-like particles, which can be fused with other functional protein fragments to form virus-like particle-like fusion proteins, greatly increasing the effect of functional fragments. Based on this mechanism, the present invention fuses the M1 protein with the HA protein fragment, and the fusion protein forms a virus-like particle, and its protective effect on avian influenza virus is much better than that of the HA protein fragment itself. The invention has great value for the development of vaccines. The invention has great value for the prevention or treatment of bird flu virus.
附图说明Description of drawings
图1为实施例1中的SDS-PAGE电泳图。Fig. 1 is the SDS-PAGE electrophoresis picture in embodiment 1.
图2为实施例1中电镜观察的照片。Fig. 2 is the photo of electron microscope observation among the embodiment 1.
图3为实施例2中的SDS-PAGE电泳图。Fig. 3 is the SDS-PAGE electrophoresis picture in embodiment 2.
图4为实施例2中电镜观察的照片。Fig. 4 is the photo of electron microscope observation among the embodiment 2.
具体实施方式detailed description
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.
大肠杆菌BL21：北京博凌科为生物科技有限公司，货号CC0503。pET-32a(+)质粒：北京诺博莱德科技有限公司。Escherichia coli BL21: Beijing Boling Kewei Biotechnology Co., Ltd., product number CC0503. pET-32a(+) plasmid: Beijing Noble Technology Co., Ltd.
禽H9N2亚型流感病毒（AvianH9N2InfluenzaAVirus）AIV-SD株，已于2011年3月22日保藏于中国微生物菌种保藏管理委员会普通微生物中心（简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号），保藏号为CGMCCNo.4689。禽H9N2亚型流感病毒又称H9N2亚型禽流感病毒。禽H9N2亚型流感病毒（AvianH9N2InfluenzaAVirus）AIV-SD株CGMCCNo.4689简称AIV-SD株。The Avian H9N2 Influenza AVirus AIV-SD strain was deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms (CGMCC for short) on March 22, 2011. The address is: No. 1 Beichen West Road, Chaoyang District, Beijing Court No. 3), the preservation number is CGMCCNo.4689. Avian H9N2 subtype influenza virus is also called H9N2 subtype avian influenza virus. Avian H9N2 subtype influenza virus (AvianH9N2InfluenzaAVirus) AIV-SD strain CGMCCNo.4689 is referred to as AIV-SD strain.
实施例1、M1蛋白的制备Embodiment 1, the preparation of M1 protein
一、重组质粒的制备1. Preparation of recombinant plasmids
1、合成序列表的序列2所示的双链DNA分子（表达序列表的序列1所示的M1蛋白）。1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing (express the M1 protein shown in sequence 1 of the sequence listing).
2、以步骤1合成的DNA分子为模板，用Primer-M1-F和Primer-M1-R组成的引物对进行PCR扩增，得到PCR扩增产物。2. Using the DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair consisting of Primer-M1-F and Primer-M1-R to obtain a PCR amplification product.
Primer-M1-F：5’-GGAATTCCATATGAGTCTTCTAACCG-3’；Primer-M1-F: 5'-GGAATTCCATATG AGTCTTCTAACCG-3';
Primer-M1-R：5’-CCGCTCGAGTACATTCTTTTCTAGT-3’。Primer-M1-R: 5'-CCGCTCGAG TACATTCTTTTTCTAGT-3'.
3、用限制性内切酶NdeⅠ和XhoⅠ双酶切步骤2的PCR扩增产物，回收酶切产物。3. The PCR amplified product of step 2 was double-digested with restriction endonucleases NdeI and XhoI, and the digested product was recovered.
4、用限制性内切酶NdeⅠ和XhoⅠ双酶切pET-32a(+)质粒，回收约5400bp的载体骨架。4. Digest the pET-32a(+) plasmid with restriction endonucleases NdeI and XhoI, and recover the vector backbone of about 5400bp.
5、将步骤3的酶切产物和步骤4的载体骨架连接，得到重组质粒甲。根据测序结果，对重组质粒甲进行结构描述如下：在pET-32a(+)质粒的NdeⅠ和XhoⅠ酶切位点之间插入了序列表的序列2自5’末端第4-756位核苷酸所示的双链DNA分子。重组质粒甲中，插入的DNA分子与pET-32a(+)质粒上的部分DNA融合，形成序列表的序列4所示的融合基因，表达序列表的序列3所示的融合蛋白（序列3中，最后6个氨基酸残基组成组氨酸标签）。5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain recombinant plasmid A. According to the sequencing results, the structure of the recombinant plasmid A is described as follows: between the NdeI and XhoI restriction sites of the pET-32a (+) plasmid, the sequence 2 of the sequence table is inserted from the 4th to the 756th nucleotide at the 5' end Double-stranded DNA molecules are shown. In the recombinant plasmid A, the inserted DNA molecule is fused with part of the DNA on the pET-32a (+) plasmid to form a fusion gene shown in sequence 4 of the sequence listing, expressing a fusion protein shown in sequence 3 of the sequence listing (in sequence 3 , the last 6 amino acid residues make up the histidine tag).
二、M1蛋白溶液的制备2. Preparation of M1 protein solution
1、将步骤一得到的重组质粒甲导入大肠杆菌BL21，得到重组菌。1. Introduce the recombinant plasmid A obtained in step 1 into Escherichia coli BL21 to obtain recombinant bacteria.
2、将步骤1得到的重组菌接种至10ml含50μg/ml氨苄青霉素的LB液体培养基，37℃、200rpm振荡培养6-8h，得到种子液。2. Inoculate 10 ml of LB liquid medium containing 50 μg/ml ampicillin with the recombinant bacteria obtained in step 1, shake and culture at 37° C. and 200 rpm for 6-8 hours to obtain seed liquid.
3、将步骤2得到的种子液接种至1000ml含50μg/ml氨苄青霉素的LB液体培养基，37℃、200rpm振荡培养，OD600nm=1时加入IPTG并使其浓度为0.1mg/ml，继续37℃、200rpm振荡培养4h。3. Inoculate the seed solution obtained in step 2 into 1000ml LB liquid medium containing 50μg/ml ampicillin, culture at 37°C and 200rpm with shaking, add IPTG when OD600nm = 1 and make the concentration 0.1mg/ml, continue for 37 Cultivate with shaking at 200rpm for 4h.
4、取步骤3的培养体系，离心收集菌体。4. Take the culture system in step 3, and collect the bacteria by centrifugation.
5、用Bindingbuffer重悬步骤4获得的菌体，8000rpm离心10min，收集菌体。5. Resuspend the bacterial cells obtained in step 4 with Bindingbuffer, centrifuge at 8000rpm for 10min, and collect the bacterial cells.
6、用Bindingbuffer重悬步骤5得到的菌体，超声破碎（超声破碎仪购自宁波新芝生物科技股份有限公司，型号为SCIENTZ-ⅡD；超声探头为10Φ；超声6s，间歇12s，超声100次），4℃、12000rpm离心15min，收集上清液。6. Resuspend the bacteria obtained in step 5 with Bindingbuffer, and ultrasonically break (the ultrasonic breaker was purchased from Ningbo Xinzhi Biotechnology Co., Ltd., the model is SCIENTZ-ⅡD; the ultrasonic probe is 10Φ; ultrasonic 6s, intermittent 12s, ultrasonic 100 times ), centrifuge at 12000rpm for 15min at 4°C, and collect the supernatant.
7、将步骤6得到的上清液上样于镍柱（购自广州誉维生物科技仪器有限公司，货号为7324610），4℃静置孵育1-2h，然后打开镍柱底部的塞子，弃除穿透液；然后用30个柱体积（实际应用中，20-50个柱体积均可）的WashBuffer洗涤镍柱，以除去非特异性结合的杂蛋白；然后用2个柱体积（实际应用中，1-3个柱体积均可）的ElutionBuffer洗脱，收集过柱后溶液，即为M1蛋白溶液。7. Load the supernatant obtained in step 6 onto a nickel column (purchased from Guangzhou Yuwei Biotechnology Instrument Co., Ltd., product number 7324610), and incubate at 4°C for 1-2 hours, then open the plug at the bottom of the nickel column, discard Remove the breakthrough solution; then use 30 column volumes (in practical applications, 20-50 column volumes are acceptable) to wash the nickel column to remove non-specifically bound impurities; then wash the nickel column with 2 column volumes (in practical applications, , 1-3 column volumes are acceptable) ElutionBuffer elution, collect the post-column solution, which is the M1 protein solution.
纯化过程中的SDS-PAGE电泳图见图1，泳道1为加入IPTG前的菌体的破碎液，泳道2为步骤6中超声破碎后得到的上清液，泳道3和泳道4为步骤7得到的M1蛋白溶液。The SDS-PAGE electrophoresis diagram during the purification process is shown in Figure 1. Lane 1 is the disrupted solution of the bacteria before adding IPTG, lane 2 is the supernatant obtained after ultrasonic crushing in step 6, and lanes 3 and 4 are obtained in step 7. M1 protein solution.
Bindingbuffer：含20mMTris、500mMNaCl、20mMimidazole，pH8.0。Binding buffer: containing 20mM Tris, 500mM NaCl, 20mMimidazole, pH8.0.
WashBuffer：含20mMTris、500mMNaCl、60mMimidazole，pH8.0。WashBuffer: Contains 20mM Tris, 500mM NaCl, 60mMimidazole, pH8.0.
ElutionBuffer：含20mMTris、500mMNaCl、500mMimidazole，pH8.0。ElutionBuffer: containing 20mM Tris, 500mM NaCl, 500mMimidazole, pH8.0.
三、对照溶液的制备3. Preparation of control solution
用pET-32a(+)质粒代替重组质粒甲进行步骤二，得到对照溶液。Use the pET-32a(+) plasmid instead of the recombinant plasmid A to perform step 2 to obtain a control solution.
四、将步骤二制备的M1蛋白溶液4℃静置12小时，然后用电镜观察形态，照片见图2。结果表明，M1蛋白可以形成病毒样颗粒。所以M1蛋白可以和功能蛋白（如具有疫苗作用的功能蛋白）形成存在形式为病毒样颗粒状的融合蛋白，该融合蛋白的效果将优于单独的功能蛋白。4. Let the M1 protein solution prepared in step 2 stand at 4°C for 12 hours, and then observe the morphology with an electron microscope. The photo is shown in Figure 2. The results showed that the M1 protein can form virus-like particles. Therefore, the M1 protein can form a fusion protein with a functional protein (such as a functional protein with a vaccine effect) in the form of a virus-like particle, and the effect of the fusion protein will be better than that of a single functional protein.
五、将步骤三制备的对照溶液4℃静置12小时，然后用电镜观察形态，观察不到病毒样颗粒。5. The control solution prepared in step 3 was left to stand at 4° C. for 12 hours, and then the morphology was observed with an electron microscope, and no virus-like particles were observed.
实施例2、融合蛋白的制备Embodiment 2, the preparation of fusion protein
一、重组质粒的构建1. Construction of recombinant plasmids
1、合成序列表的序列6自5’末端第1至801位所示的双链DNA分子(表达序列表的序列5自N端第1至267位核苷酸所示的蛋白质，序列5中，第1至252位氨基酸残基组成M1蛋白，第253至267位氨基酸残基组成HA蛋白片段)。1. Synthesize the double-stranded DNA molecule shown in the sequence 6 of the sequence listing from the 1st to the 801st position of the 5' end (express the protein shown in the sequence 5 of the sequence listing from the 1st to the 267th nucleotide of the N terminal, in the sequence 5 , amino acid residues 1 to 252 constitute the M1 protein, and amino acid residues 253 to 267 constitute the HA protein fragment).
2、以步骤1合成的DNA分子为模板，用Primer-F和Primer-R组成的引物对进行PCR扩增，得到PCR扩增产物。2. Using the DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of Primer-F and Primer-R to obtain a PCR amplification product.
Primer-F：5’-GGAATTCCATATGAGTCTTCTAACCG-3’；Primer-F: 5'-GGAATTCCATATG AGTCTTCTAACCG-3';
Primer-R：5’-CCGCTCGAGCAGCTTGGGGTAGCT-3’。Primer-R: 5'-CCGCTCGAG CAGCTTGGGGTAGCT-3'.
3、用限制性内切酶NdeⅠ和XhoⅠ双酶切步骤2的PCR扩增产物，回收酶切产物。3. The PCR amplified product of step 2 was double-digested with restriction endonucleases NdeI and XhoI, and the digested product was recovered.
4、用限制性内切酶NdeⅠ和XhoⅠ双酶切pET-32a(+)质粒，回收约5400bp的载体骨架。4. Digest the pET-32a(+) plasmid with restriction endonucleases NdeI and XhoI, and recover the vector backbone of about 5400bp.
5、将步骤3的酶切产物和步骤4的载体骨架连接，得到重组质粒乙。根据测序结果，对重组质粒乙进行结构描述如下：在pET-32a(+)质粒的NdeⅠ和XhoⅠ酶切位点之间插入了序列表的序列6自5’末端第4-801位核苷酸所示的双链DNA分子。重组质粒乙中，插入的DNA分子与pET-32a(+)质粒上的部分DNA融合，形成序列表的序列6所示的融合基因，表达序列表的序列5所示的融合蛋白（序列5中，最后6个氨基酸残基组成组氨酸标签）。5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain recombinant plasmid B. According to the sequencing results, the structure of the recombinant plasmid B is described as follows: between the NdeI and XhoI restriction sites of the pET-32a(+) plasmid, the sequence 6 of the sequence table is inserted from the 4th to the 801st nucleotide at the 5' end Double-stranded DNA molecules are shown. In the recombinant plasmid B, the inserted DNA molecule is fused with part of the DNA on the pET-32a (+) plasmid to form a fusion gene shown in sequence 6 of the sequence listing, expressing a fusion protein shown in sequence 5 of the sequence listing (in sequence 5 , the last 6 amino acid residues make up the histidine tag).
二、融合蛋白溶液的制备2. Preparation of fusion protein solution
用重组质粒乙代替重组质粒甲，其它同实施例1的步骤二。Recombinant plasmid B is used to replace recombinant plasmid A, and other steps are the same as step 2 of Example 1.
得到融合蛋白溶液。Obtain fusion protein solution.
纯化过程中的SDS-PAGE电泳图见图3，泳道1为加入IPTG前的菌体的破碎液，泳道2为步骤6中超声破碎后得到的上清液，泳道3为步骤7得到的融合蛋白溶液。The SDS-PAGE electrophoresis image during the purification process is shown in Figure 3. Lane 1 is the disrupted liquid of the bacteria before adding IPTG, lane 2 is the supernatant obtained after ultrasonic disruption in step 6, and lane 3 is the fusion protein obtained in step 7. solution.
三、HA片段溶液的制备3. Preparation of HA fragment solution
将序列表的序列6自5’末端第757至801位核苷酸所述的双链DNA分子插入pET-32a(+)质粒的NdeⅠ和XhoⅠ酶切位点之间，得到重组质粒丙。Insert the double-stranded DNA molecule described in the 757th to 801st nucleotides from the 5' end of sequence 6 in the sequence listing between the NdeI and XhoI restriction sites of the pET-32a (+) plasmid to obtain the recombinant plasmid C.
用重组质粒丙代替重组质粒乙，其它同实施例1的步骤二，得到HA片段溶液。Recombinant plasmid C was used to replace recombinant plasmid B, and other steps were the same as step 2 of Example 1 to obtain HA fragment solution.
四、对照溶液的制备Fourth, the preparation of the control solution
用pET-32a(+)质粒代替重组质粒乙，其它同实施例1的步骤二，得到对照溶液。The pET-32a(+) plasmid was used to replace the recombinant plasmid B, and the others were the same as step 2 of Example 1 to obtain a control solution.
五、将步骤二制备的融合蛋白溶液4℃静置12小时，然后用电镜观察形态，照片见图4。结果表明，融合蛋白可以形成病毒样颗粒。5. Let the fusion protein solution prepared in step 2 stand at 4°C for 12 hours, and then observe its morphology with an electron microscope. The photo is shown in Figure 4. The results showed that the fusion proteins could form virus-like particles.
六、将步骤三制备的HA片段溶液4℃静置12小时，然后用电镜观察形态，观察不到病毒样颗粒。6. The HA fragment solution prepared in step 3 was left to stand at 4° C. for 12 hours, and then the morphology was observed with an electron microscope, and no virus-like particles were observed.
七、将步骤四制备的对照溶液4℃静置12小时，然后用电镜观察形态，观察不到病毒样颗粒。7. Let the control solution prepared in step 4 stand at 4° C. for 12 hours, and then observe the morphology with an electron microscope, and no virus-like particles can be observed.
实施例3、疫苗的制备Embodiment 3, the preparation of vaccine
一、疫苗甲的制备1. Preparation of Vaccine A
油相：弗氏佐剂（购自上海博蕴生物科技有限公司代理sigma产品，货号：BY12096）。Oil phase: Freund's adjuvant (purchased from Shanghai Boyun Biotechnology Co., Ltd. as an agent of sigma products, item number: BY12096).
水相：实施例2制备的融合蛋白溶液（蛋白浓度为0.1mg/ml）。Water phase: the fusion protein solution prepared in Example 2 (protein concentration: 0.1 mg/ml).
乳化：取1体积份油相放于乳化缸内，开动电机慢速转动搅拌，同时缓慢加入1体积份水相，加完后再以10000转/分钟，搅拌2～5分钟；得到疫苗甲。Emulsification: Take 1 volume part of the oil phase and put it in the emulsification tank, start the motor to rotate and stir at a slow speed, and at the same time slowly add 1 volume part of the water phase, and then stir at 10000 rpm for 2 to 5 minutes to obtain Vaccine A.
二、疫苗乙的制备2. Preparation of Vaccine B
用实施例2制备的HA片段溶液等体积代替融合蛋白溶液进行步骤一，得到疫苗乙。The HA fragment solution prepared in Example 2 was used to replace the fusion protein solution in an equal volume to carry out Step 1 to obtain Vaccine B.
三、对照品的制备3. Preparation of reference substance
用实施例2制备的对照溶液等体积代替融合蛋白溶液进行步骤一，得到对照品。Use the same volume of the control solution prepared in Example 2 to replace the fusion protein solution to carry out step 1 to obtain the reference substance.
实施例4、融合蛋白的应用Embodiment 4, the application of fusion protein
一、对雏鸡免疫的效力1. Efficacy of immunity to chicks
将雏鸡(品种为SPF白杭鸡，购自北京维通利华实验动物技术有限公司)分为6组，每组10只，分别进行如下处理：The chicks (the breed is SPF Baihang chicken, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were divided into 6 groups, 10 in each group, and were processed as follows:
第一组：单次注射实施例3制备的疫苗甲，每只雏鸡皮下注射0.1ml；The first group: a single injection of the vaccine A prepared in Example 3, subcutaneous injection of 0.1ml per chick;
第二组：单次注射实施例3制备的疫苗甲，每只雏鸡皮下注射0.2ml；The second group: a single injection of the vaccine A prepared in Example 3, each chick was subcutaneously injected with 0.2ml;
第三组：单次注射实施例3制备的疫苗甲，每只雏鸡皮下注射0.4ml；The third group: a single injection of the vaccine A prepared in Example 3, each chick was subcutaneously injected with 0.4ml;
第四组：单次注射实施例3制备的疫苗乙，每只雏鸡皮下注射0.4ml；The fourth group: a single injection of the vaccine B prepared in Example 3, each chick was subcutaneously injected with 0.4ml;
第五组：单次注射实施例3制备的对照品，每只雏鸡皮下注射0.4ml；The fifth group: a single injection of the reference substance prepared in Example 3, subcutaneous injection of 0.4ml per chick;
第六组：单次注射生理盐水，每只雏鸡皮下注射0.1ml。The sixth group: a single injection of normal saline, 0.1ml per chick subcutaneously.
免疫后第14天，用AIV-SD株病毒液对雏鸡进行攻毒（注射雏鸡腿部肌肉），每只雏鸡的攻毒剂量为107EID50，之后再连续观察14天。攻毒后第14天统计攻毒保护数，结果见表1。On the 14th day after immunization, the chicks were challenged with the AIV-SD strain virus solution (injected into the leg muscles of the chicks), and the challenge dose of each chick was 107 EID50 , and then observed continuously for 14 days. On the 14th day after the challenge, the number of protection against the virus was counted, and the results are shown in Table 1.
表1雏鸡疫苗的效力试验结果Table 1 Efficacy test results of chick vaccines
免疫疫苗甲的实验组的保护率均大于80%，免疫剂量为0.2ml和0.4ml时，保护率达到100%。免疫疫苗乙的实验组的保护率为60%。免疫对照品的实验组的保护率为0%。免疫生理盐水的实验组的保护率为0%。The protection rate of the experimental group immunized with vaccine A was greater than 80%, and the protection rate reached 100% when the immunization dose was 0.2ml and 0.4ml. The experimental group immunized with vaccine B had a protection rate of 60%. The protection rate of the experimental group of the immune control substance was 0%. The protection rate of the experimental group immunized with physiological saline was 0%.
以上数据均表明,融合蛋白可以作为疫苗使用，对禽流感起到良好的保护预防作用，可以有效地预防鸡群禽流感的发生，对鸡群具有保护力。The above data all show that the fusion protein can be used as a vaccine, which has a good protective and preventive effect on avian influenza, can effectively prevent the occurrence of avian influenza in chicken flocks, and has protection for chicken flocks.
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