技术领域technical field
本发明属于基因工程学，免疫学和肿瘤学，涉及一种重组溶瘤痘苗病毒及其制备方法与应用。The invention belongs to genetic engineering, immunology and oncology, and relates to a recombinant oncolytic vaccinia virus and its preparation method and application.
背景技术Background technique
恶性淋巴瘤的发病率占整个癌症患者的3-5％，每年有452,000新增患者。大于90％的成人患者来源于成熟的B细胞，提示B细胞淋巴肿瘤是威胁人类健康的重要疾病之一。The incidence of malignant lymphoma accounts for 3-5% of all cancer patients, with 452,000 new patients every year. More than 90% of adult patients are derived from mature B cells, suggesting that B cell lymphoma is one of the important diseases that threaten human health.
免疫治疗是继手术，放疗，化疗，分子靶向治疗后又一种癌症治疗新方法。在各种癌症免疫治疗方法中，免疫检查点抑制剂是近年来获得突出疗效的药物之一。免疫检查点是预防免疫系统过度激活的抑制性通路，在被激活的免疫细胞表面存在一些蛋白如CTLA4，PD1等在过度免疫反应时起到“刹车”作用，然而在肿瘤发生时，这些蛋白因过度表达而持久地为免疫系统发出抑制信号，从而促进肿瘤的免疫逃逸。故免疫检查点抑制剂在癌症的免疫治疗中起着重要的作用。Immunotherapy is another new method of cancer treatment after surgery, radiotherapy, chemotherapy, and molecular targeted therapy. Among various cancer immunotherapy methods, immune checkpoint inhibitors are one of the drugs that have achieved outstanding efficacy in recent years. Immune checkpoints are inhibitory pathways that prevent excessive activation of the immune system. There are some proteins such as CTLA4 and PD1 on the surface of activated immune cells that act as "brakes" during excessive immune responses. However, when tumors occur, these proteins are due to Overexpression and persistently send an inhibitory signal to the immune system, thereby promoting immune escape of tumors. Therefore, immune checkpoint inhibitors play an important role in cancer immunotherapy.
PD-1/PD-L1信号通路是阻断免疫检查点策略的最为重要的靶点之一。在肿瘤发生时可导致T细胞表达PD1，多种类型癌症的肿瘤侵润淋巴细胞(TIL)也表达PD-1。同时研究表明多种癌细胞高表达PD-L1，包括淋巴瘤，白血病，恶性黑色素瘤和非小细胞肺癌(NSCLC)等。PD-1与癌细胞表面的PD-1配体(PD-L1或者PD-L2)结合抑制T细胞的活化，导致特异性T细胞耗竭或失能。The PD-1/PD-L1 signaling pathway is one of the most important targets for blocking immune checkpoint strategies. T cells can express PD1 during tumorigenesis, and tumor infiltrating lymphocytes (TIL) of various types of cancer also express PD-1. At the same time, studies have shown that a variety of cancer cells highly express PD-L1, including lymphoma, leukemia, malignant melanoma and non-small cell lung cancer (NSCLC). The combination of PD-1 and the PD-1 ligand (PD-L1 or PD-L2) on the surface of cancer cells inhibits the activation of T cells, resulting in the exhaustion or failure of specific T cells.
在B细胞淋巴肿瘤上，研究显示滤泡淋巴瘤(FL)的TIL和外周血T细胞上高表达PD1，且T细胞失能。另一方面，多种B细胞肿瘤，包括弥漫大B淋巴瘤(DLBCL)，原发纵膈大B淋巴瘤(PMBCL)以及睾丸大B淋巴瘤上均大量表达PD-L1。因此，有效地阻断PD-1/PD-L1信号通路，能够增强T细胞对B细胞淋巴肿瘤的杀伤。In B-cell lymphoid tumors, studies have shown that PD1 is highly expressed on TILs and peripheral blood T cells of follicular lymphoma (FL), and T cells are disabled. On the other hand, a variety of B cell tumors, including diffuse large B lymphoma (DLBCL), primary mediastinal large B lymphoma (PMBCL) and testicular large B lymphoma, all express PD-L1 in large quantities. Therefore, effectively blocking the PD-1/PD-L1 signaling pathway can enhance the killing of T cells against B-cell lymphoid tumors.
新近，阻断免疫检查点的PD-1和PD-L1单抗和抑制剂已应用于I-III期临床研究。2015年6月，美国食品及药物管理局(FDA)批准PD-1单抗nivolumab作为一款治疗药物用于先前经过化疗的晚期NSCLC患者。I期临床研究表明，nivolumab治疗NSCLC患者的平均总生存期为14.9个月，1年和3年的生存率为42％和18％。Recently, PD-1 and PD-L1 monoclonal antibodies and inhibitors that block immune checkpoints have been applied in phase I-III clinical studies. In June 2015, the US Food and Drug Administration (FDA) approved the PD-1 monoclonal antibody nivolumab as a treatment drug for patients with advanced NSCLC who have previously undergone chemotherapy. Phase I clinical studies have shown that the average overall survival of NSCLC patients treated with nivolumab was 14.9 months, and the 1-year and 3-year survival rates were 42% and 18%.
但是，免疫检查点抑制剂也有其局限性：(1)多数病人不能获得持久的完全反应；(2)PD-1单抗治疗后约25-30％患者出现严重免疫相关副作用(irAEs)；(3)PD-1/PD-L1通路在肿瘤微环境中的特点影响了PD-1单抗的疗效。联合治疗是克服免疫检查点抑制剂局限性的主要研究方向。目前正在研究的策略包括抗PD-1单抗与放化疗、小分子靶向药物、其他免疫检查点抑制剂或溶瘤病毒联合。However, immune checkpoint inhibitors also have their limitations: (1) Most patients cannot achieve a durable complete response; (2) About 25-30% of patients experience severe immune-related side effects (irAEs) after PD-1 monoclonal antibody treatment; ( 3) The characteristics of the PD-1/PD-L1 pathway in the tumor microenvironment affect the efficacy of PD-1 monoclonal antibody. Combination therapy is a major research direction to overcome the limitations of immune checkpoint inhibitors. Strategies currently being investigated include anti-PD-1 mAbs in combination with chemoradiotherapy, small molecule targeted agents, other immune checkpoint inhibitors, or oncolytic viruses.
溶瘤病毒(Oncolytic virus，OV)疗法是今年来发展迅速的一种肿瘤生物治疗新方法。2015年10月，FDA批准安进公司的OV产品T-Vec用于治疗恶性黑色素瘤。12月，T-Vec又获批欧洲食品药品管理局，证明OV疗法是治疗癌症的一种非常有前景的新方法。溶瘤痘苗病毒(Oncolytic vaccinia virus,OVV)是近年来备受关注的一类溶瘤病毒。其优点在于：病毒稳定性好、致病性低、基因转染效力高、安全性良好。溶瘤痘苗病毒能选择性感染肿瘤细胞并在细胞内复制、杀死肿瘤细胞，而对正常组织和细胞的毒性很小。JX-594是第一个用于临床研究的溶瘤痘苗病毒，它缺失了胸腺嘧啶核苷激酶(TK)区并携带外源性基因GM-CSF，JX-594能在快速增殖的肿瘤细胞中复制，同时携带的GM-CSF能进一步刺激抗肿瘤免疫。Breitbach CJ等里程碑式的研究结果显示：JX-594静脉注射23例难治、复发的实体肿瘤，13例患者有效，1例患者获得部分缓解；表明OVV可以用于静脉注射，发挥全身抗肿瘤作用。Oncolytic virus (OV) therapy is a new method of tumor biological treatment that has developed rapidly this year. In October 2015, the FDA approved Amgen's OV product T-Vec for the treatment of malignant melanoma. In December, T-Vec was approved by the European Food and Drug Administration, proving that OV therapy is a very promising new method for treating cancer. Oncolytic vaccinia virus (OVV) is a type of oncolytic virus that has attracted much attention in recent years. The advantages are: good virus stability, low pathogenicity, high gene transfection efficiency and good safety. Oncolytic vaccinia virus can selectively infect tumor cells, replicate in cells, and kill tumor cells with little toxicity to normal tissues and cells. JX-594 is the first oncolytic vaccinia virus used in clinical research. It has deleted the thymidine kinase (TK) region and carries the exogenous gene GM-CSF. JX-594 can be used in rapidly proliferating tumor cells Replication, while carrying GM-CSF can further stimulate anti-tumor immunity. The landmark research results of Breitbach CJ and others showed that: JX-594 was injected intravenously in 23 cases of refractory and recurrent solid tumors, 13 patients were effective, and 1 patient achieved partial remission; indicating that OVV can be used for intravenous injection to exert systemic anti-tumor effect .
发明内容Contents of the invention
本发明的发明人发现，现有的PD1的单抗在治疗B细胞淋巴肿瘤中存在局限性。为此，本发明提供一种重组溶瘤痘苗病毒及其制备方法与应用。The inventors of the present invention found that the existing PD1 monoclonal antibody has limitations in the treatment of B-cell lymphoma. Therefore, the present invention provides a recombinant oncolytic vaccinia virus and its preparation method and application.
本发明的目的是通过以下技术方案实现的：一种重组溶瘤痘苗病毒，所述病毒的胸腺嘧啶核苷激酶(TK)区包含SEQ ID NO.1所示的PD1全长抗体的编码序列。The object of the present invention is achieved through the following technical solutions: a recombinant oncolytic vaccinia virus, the thymidine kinase (TK) region of the virus contains the coding sequence of the PD1 full-length antibody shown in SEQ ID NO.1.
一种重组溶瘤痘苗病毒的制备方法，其特征在于，包括以下步骤：A method for preparing a recombinant oncolytic vaccinia virus, comprising the following steps:
(1)合成人全长PD-1单克隆抗体，其基因序列如SEQ ID NO.1所示；(1) Synthesize human full-length PD-1 monoclonal antibody, whose gene sequence is shown in SEQ ID NO.1;
(2)扩增全长PD-1单克隆抗体，酶切后亚克隆至痘苗病毒穿梭质粒(pTK)的TK区中，构建出重组质粒pCB-aPD1。其中，aPD1基因由痘苗病毒的早/晚期启动子(P-se/l)控制。(2) The full-length PD-1 monoclonal antibody was amplified, and subcloned into the TK region of the vaccinia virus shuttle plasmid (pTK) after enzyme digestion to construct the recombinant plasmid pCB-aPD1. Among them, the aPD1 gene is controlled by the early/late promoter (P-se/l) of vaccinia virus.
(3)采用基因同源重组的方式，将pCB-aPD1质粒转染到已经被感染了野生型痘苗病毒的细胞中，使两者同源重组，产生重组痘苗病毒VV-aPD1。经筛选后，获得所述TK区包含SEQ ID NO.1所示的PD1全长抗体的编码序列的重组溶瘤痘苗病毒。(3) Transfect the pCB-aPD1 plasmid into cells infected with wild-type vaccinia virus by means of gene homologous recombination, so that the two are homologously recombined to produce recombinant vaccinia virus VV-aPD1. After screening, the recombinant oncolytic vaccinia virus whose TK region contains the coding sequence of the PD1 full-length antibody shown in SEQ ID NO.1 is obtained.
一种重组溶瘤痘苗病毒在制备治疗治疗B细胞淋巴肿瘤的药物中的应用。The application of a recombinant oncolytic vaccinia virus in the preparation of a drug for treating B-cell lymphoma.
本发明的有益效果是：The beneficial effects of the present invention are:
1、本发明将基因治疗的抑瘤效果与病毒治疗的溶瘤效应有效结合起来，制备了一种可高效表达PD1全长抗体基因的溶瘤痘苗病毒。在溶瘤病毒发挥溶瘤效应裂解肿瘤细胞的同时，大量表达PD1全长抗体，抑制T细胞表面PD1的活性，激活T细胞免疫应答，发挥双重的抗肿瘤效果。相对于单纯的基因疗法或者病毒疗法增强了其对恶性B细胞淋巴肿瘤的杀伤能力。1. The present invention effectively combines the tumor-suppressing effect of gene therapy with the oncolytic effect of virus therapy, and prepares an oncolytic vaccinia virus that can efficiently express the PD1 full-length antibody gene. While the oncolytic virus exerts its oncolytic effect to lyse tumor cells, it also expresses a large amount of full-length PD1 antibody, inhibits the activity of PD1 on the surface of T cells, activates the immune response of T cells, and exerts a dual anti-tumor effect. Compared with simple gene therapy or virus therapy, it has enhanced its ability to kill malignant B cell lymphoma.
2、本发明采用病毒复制相关基因缺失的方式，缺失痘苗病毒基因组的TK区，确保病毒在异常增殖的肿瘤细胞内特异性复制，而在正常的细胞中则不能复制，大大增强了溶瘤痘苗病毒载体的安全性。2. The present invention adopts the method of virus replication-related gene deletion, and deletes the TK region of the vaccinia virus genome to ensure that the virus replicates specifically in abnormally proliferating tumor cells, but cannot replicate in normal cells, greatly enhancing the oncolytic vaccinia. Safety of viral vectors.
附图说明Description of drawings
图1a是pCB-aPD-1经ECORI和BglII双酶切后DNA凝胶电泳结果，1b是部分测序结果。Figure 1a is the DNA gel electrophoresis result of pCB-aPD-1 after double digestion with ECORI and BglII, and 1b is the partial sequencing result.
图2是流式检测pCB-aPD-1与活化的T细胞孵育后的PD-1表达，继而检测PD-1抗体活性，其中，左图为未经刺激的Jurkat细胞对照，右图为经ConA刺激后表达PD1抗原的Jurkat细胞。Figure 2 is flow cytometric detection of PD-1 expression after incubation of pCB-aPD-1 and activated T cells, and then detection of PD-1 antibody activity, in which, the left picture is the unstimulated Jurkat cell control, and the right picture is ConA Jurkat cells expressing PD1 antigen after stimulation.
图3是pCB-aPD1-GFP与WT在293A细胞中同源重组后的荧光图。Fig. 3 is a fluorescent image of pCB-aPD1-GFP and WT after homologous recombination in 293A cells.
图4是OVV-aPD-1重组后，提取病毒基因组DNA通过PCR检测目的基因和野毒基因的存在。Fig. 4 shows that after OVV-aPD-1 recombination, the viral genomic DNA was extracted to detect the presence of the target gene and the wild virus gene by PCR.
图5是OVV-aPD-1以浓度梯度感染Raji细胞株后，流式检测GFP的表达效率。Figure 5 shows the expression efficiency of GFP detected by flow cytometry after OVV-aPD-1 infected Raji cell line with a concentration gradient.
图6是OVV-aPD-1以浓度梯度感染Raji细胞株后，通过检测集落形成和CCK8染色法检测OVV-aPD-1的细胞毒性，其中，b是对a的数据分析。Figure 6 shows the cytotoxicity of OVV-aPD-1 detected by detecting colony formation and CCK8 staining after OVV-aPD-1 was infected with a concentration gradient of Raji cell line, where b is the data analysis of a.
具体实施方式Detailed ways
本发明结合附图和实例作进一步的说明。除特别说明外，本发明可采用本领域常规技术。The present invention is further described in conjunction with accompanying drawings and examples. Unless otherwise specified, the present invention may adopt conventional techniques in the art.
1、人全长PD-1单克隆抗体的合成：1. Synthesis of human full-length PD-1 monoclonal antibody:
PD-1单克隆抗体的单链可变区序列参照施贵宝PD-1单抗nivolumab的序列，通过氨基酸推演和密码子优化得到我们设计的PD-1单链可变区序列，单抗的Fc段参照人IgG4的Fc段序列，采用化学合成的方法合成人PD-1抗体的重链和轻链，该重链和轻链基因由链接子IRES连接。PD-1单克隆抗体的全长基因序列如SEQ ID NO.1所示。The sequence of the single-chain variable region of the PD-1 monoclonal antibody refers to the sequence of Bristol-Myers Squibb’s PD-1 monoclonal antibody nivolumab, and the sequence of the PD-1 single-chain variable region designed by us is obtained through amino acid deduction and codon optimization. The Fc segment of the monoclonal antibody Referring to the Fc segment sequence of human IgG4, the heavy chain and light chain of human PD-1 antibody were synthesized by chemical synthesis, and the heavy chain and light chain genes were linked by the linker IRES. The full-length gene sequence of the PD-1 monoclonal antibody is shown in SEQ ID NO.1.
2、携带人PD-1单抗的溶瘤痘苗病毒穿梭质粒pCB-aPD1的构建与鉴定：2. Construction and identification of oncolytic vaccinia virus shuttle plasmid pCB-aPD1 carrying human PD-1 monoclonal antibody:
应用末端含有ECORI和BglII酶切位点的特异性PCR引物扩增出PD1全长抗体序列(含重链和轻链)，再经过ECORI和BglII双酶切后亚克隆至携带GFP基因的痘苗病毒穿梭质粒(pTK-GFP)的TK区中，构建出pCB-aPD1。酶切后的DNA凝胶电泳结果如图1a所示，从图中可以看出pCB-aPD1经酶切后产生2700bp的aPD1全长序列；1b是部分测序结果，结果证明所克隆的aPD1基因序列正确。aPD1基因由痘苗病毒的早/晚期启动子(P-se/l)控制。构建出的阳性克隆应用酶切、测序进行鉴定，选择鉴定正确的克隆进行质粒大量抽提，制备>4μg的高纯度质粒，-80℃保存备用。The PD1 full-length antibody sequence (including heavy chain and light chain) was amplified using specific PCR primers containing ECORI and BglII restriction sites at the end, and then subcloned into vaccinia virus carrying the GFP gene after double digestion with ECORI and BglII In the TK region of the shuttle plasmid (pTK-GFP), pCB-aPD1 was constructed. The results of DNA gel electrophoresis after enzyme digestion are shown in Figure 1a. It can be seen from the figure that pCB-aPD1 produces a 2700bp full-length sequence of aPD1 after enzyme digestion; 1b is the partial sequencing result, which proves that the cloned aPD1 gene sequence correct. The aPD1 gene is controlled by the early/late promoter (P-se/l) of vaccinia virus. The constructed positive clones should be identified by enzyme digestion and sequencing, and the correctly identified clones should be selected for large-scale extraction of plasmids to prepare >4 μg of high-purity plasmids and stored at -80°C for later use.
其中，痘苗病毒穿梭质粒可参考房有荣,李红玉,陈科达,周文硕,&阎辉.(2012).Zeocin和gfp双筛选标记重组痘苗病毒载体的构建.国际流行病学传染病学杂志,39(3),148-152。Among them, the vaccinia virus shuttle plasmid can refer to Fang Yourong, Li Hongyu, Chen Keda, Zhou Wenshuo, & Yan Hui. (2012). Construction of recombinant vaccinia virus vector with Zeocin and gfp dual selection markers. International Journal of Epidemiology and Infectious Diseases , 39(3), 148-152.
3、pCB-aPD-1表达PD-1抗体的检测3. Detection of pCB-aPD-1 expressing PD-1 antibody
Jurkat细胞经ConA刺激后转导pCB-aPD-1，应用PE-anti Human IgG4(Fc)通过流式检测pCB-aPD-1表达PD-1抗体能力，结果如图2所示，说明pCB-aPD-1能表达11.9％的PD-1抗体。After Jurkat cells were stimulated by ConA, pCB-aPD-1 was transduced, and PE-anti Human IgG4 (Fc) was used to detect the ability of pCB-aPD-1 to express PD-1 antibody by flow cytometry. The results are shown in Figure 2, indicating that pCB-aPD -1 can express 11.9% of PD-1 antibodies.
4、OVV-a PD1病毒的包装与鉴定4. Packaging and identification of OVV-a PD1 virus
采用基因同源重组的方式，通过脂质体转染将pCB-aPD1-GFP质粒转染到已经被感染了野生型痘苗病毒的293A细胞中，使两者同源重组，产生VV-aPD-1-GFP。包装的病毒经3-5轮药筛(霉酚酸，黄嘌呤与雌黄嘌呤)联合GFP显影筛选，嗜斑纯化，连续至少三轮“挑斑”，PCR鉴定，筛选出正确的阳性重组痘苗病毒。图3结果说明pCB-aPD1-GFP在293A细胞里成功与野生型痘苗病毒重组，产生病毒空斑。图4结果表明经多轮药筛产生的重组病毒携带PD1全长抗体的重链(1300bp)，轻链(704bp)和链接子IRES(650bp)序列，且无野毒污染。Using gene homologous recombination, the pCB-aPD1-GFP plasmid was transfected into 293A cells that had been infected with wild-type vaccinia virus by liposome transfection, so that the two were homologously recombined to produce VV-aPD-1 -GFP. The packaged virus was screened by 3-5 rounds of drug screening (mycophenolic acid, xanthine and estraxanthine) combined with GFP imaging, purified for plaque, at least three consecutive rounds of "spot picking", PCR identification, and the correct positive recombinant vaccinia virus was screened out . The results in Figure 3 show that pCB-aPD1-GFP successfully recombined with wild-type vaccinia virus in 293A cells, resulting in viral plaques. The results in Figure 4 show that the recombinant virus produced by multiple rounds of drug screening carries the heavy chain (1300bp), light chain (704bp) and linker IRES (650bp) sequences of the PD1 full-length antibody, and has no wild virus contamination.
5、OVV-aPD1对B细胞淋巴瘤细胞株的体外感染5. In vitro infection of B-cell lymphoma cell lines by OVV-aPD1
为验证OVV-aPD1对B细胞淋巴瘤细胞株的体外感染，选取Raji细胞株，OVV-aPD1分别以5，10，20MOI的浓度梯度感染Raji细胞株，利用流式细胞仪检测OVV-aPD1对Raji细胞株的感染效率，结果如图5所示，OVV-aPD1对Raji细胞株的感染效率在20MOI时达到24.28％。In order to verify the in vitro infection of OVV-aPD1 on B-cell lymphoma cell lines, the Raji cell line was selected, and OVV-aPD1 was used to infect the Raji cell line at a concentration gradient of 5, 10, and 20 MOI respectively, and the effect of OVV-aPD1 on Raji was detected by flow cytometry. The infection efficiency of the cell line, as shown in Figure 5, the infection efficiency of OVV-aPD1 on the Raji cell line reached 24.28% at 20 MOI.
6、OVV-a PD1对B细胞淋巴瘤细胞株的体外杀伤6. In vitro killing effect of OVV-a PD1 on B-cell lymphoma cell lines
为验证OVV-a PD1对B细胞淋巴瘤细胞株的体外杀伤，OVV-aPD1分别以5，10，20MOI的浓度梯度感染Raji细胞株72小时，利用集落形成实验和CCK8染色法检测OVV-a PD1对Raji细胞株的生长抑制作用，结果如图6a和6b所示，OVV-a PD1对Raji细胞株的生长抑制作用呈浓度依赖，在20MOI时细胞呈小且分散集落生长，细胞毒性作用达到65％，严重影响细胞增殖。In order to verify the killing effect of OVV-a PD1 on B-cell lymphoma cell lines in vitro, OVV-aPD1 was used to infect Raji cell lines at a concentration gradient of 5, 10, and 20 MOI for 72 hours, and the colony formation assay and CCK8 staining were used to detect OVV-a PD1 The growth inhibitory effect on Raji cell line, the results are shown in Figure 6a and 6b, the growth inhibitory effect of OVV-a PD1 on Raji cell line is concentration-dependent, and the cells grow in small and scattered colonies at 20 MOI, and the cytotoxic effect reaches 65 %, seriously affecting cell proliferation.
序列表sequence listing
<110> 浙江大学<110> Zhejiang University
<120> 一种重组溶瘤痘苗病毒及其制备方法与应用<120> A recombinant oncolytic vaccinia virus and its preparation method and application
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<211> 2702<211> 2702
<212> DNA<212>DNA
<213> UN<213> UN
<400> 1<400> 1
gaattctcac ttgcccaggg acagggacag gcttttctga gtataatggt tgtgcagtgc 60gaattctcac ttgcccaggg acagggacag gcttttctga gtataatggt tgtgcagtgc 60
ttcgtgcatc acggagcagg agaacacgtt gccctcctgc catctagact tatccacggt 120ttcgtgcatc acggagcagg agaacacgtt gccctcctgc catctagact tatccacggt 120
cagccggcta tacagaaaga agctgccatc ggagtccagc acgggtggtg tggtcttgta 180cagccggcta tacagaaaga agctgccatc ggagtccagc acgggtggtg tggtcttgta 180
attgttctcg ggctggccat tggactccca ctccacagcg atgtcagatg gatagaagcc 240atgttctcg ggctggccat tggactccca ctccacagcg atgtcagatg gatagaagcc 240
cttcaccaga caggtcaggc tcacctggtt ctttgtcatc tcctcctggg aagggggcag 300cttcaccaga caggtcaggc tcacctggtt ctttgtcatc tcctcctggg aagggggcag 300
ggtgtacacc tgtggctccc taggctggcc cttggcctta gagattgtct tctcgatgct 360ggtgtacacc tgtggctccc taggctggcc cttggcctta gagattgtct tctcgatgct 360
ggatggcagg cccttattgc tcaccttgca cttatactcc ttgccgttca gccagtcctg 420ggatggcagg cccttattgc tcaccttgca cttatactcc ttgccgttca gccagtcctg 420
atgcagcacg gtcagcacgc tcaccacccg gtatgtggag ttaaactgct cctccctagg 480atgcagcacg gtcagcacgc tcaccacccg gtatgtggag ttaaactgct cctccctagg 480
cttggtctta gcattgtgca cctccacgcc atccacatac cagttgaact gcacctctgg 540cttggtctta gcattgtgca cctccacgcc atccacatac cagttgaact gcacctctgg 540
atcctcctgg gacacgtcca ccaccacgca ggtcacctca ggtgtccgag agatcatcag 600atcctcctgg gacacgtcca ccaccacgca ggtcacctca ggtgtccgag agatcatcag 600
tgtgtcctta ggctttggag gaaacaggaa cacggaaggt cctcccagga actcaggagc 660tgtgtcctta ggctttggag gaaacaggaa cacggaaggt cctcccagga actcaggagc 660
aggacaaggt gggcaaggag gtccgtactt agactccacc ctcttatcca ccttggtatt 720aggacaaggt gggcaaggag gtccgtactt agactccacc ctcttatcca ccttggtatt 720
gctaggctta tggtccacgt tgcaggtata tgtcttggtg cccagagagc tggagggcac 780gctaggctta tggtccacgt tgcaggtata tgtcttggtg cccagagagc tggagggcac 780
tgtcaccaca gagctcaggc tgtacaggcc ggaagactgc agcacagcag gaaaggtgtg 840tgtcaccaca gagctcaggc tgtacaggcc ggaagactgc agcacagcag gaaaggtgtg 840
cacgccggat gtcagggcgc cggagttcca gctcacggtc actggctcag ggaaataatc 900cacgccggat gtcagggcgc cggagttcca gctcacggtc actggctcag ggaaataatc 900
cttcaccaga cagcccagag cggcggtgct ctcggatgta gacctgctgc agggagccag 960cttcaccaga cagcccagag cggcggtgct ctcggatgta gacctgctgc agggagccag 960
tgggaacacg gaggggccct ttgtgctggc gctggacacg gtcaccagtg tgccctggcc 1020tgggaacacg gaggggccct ttgtgctggc gctggacacg gtcaccagtg tgccctggcc 1020
ccagtaatcg tcattggtag cacaatagta cacggctgta tcctcggccc tcagagagtt 1080ccagtaatcg tcattggtag cacaatagta cacggctgta tcctcggccc tcagagagtt 1080
catctgcaga aacagggtat tcttggagtt gtccctagag attgtgaatc tgcccttcac 1140catctgcaga aacagggtat tcttggagtt gtccctagag attgtgaatc tgcccttcac 1140
ggaatcggca tagtacctct tagagccgtc gtaccagatc acagccaccc actccagtcc 1200ggaatcggca tagtacctct tagagccgtc gtaccagatc acagccacccc actccagtcc 1200
cttgccagga gcctgtctca cccagtgcat gccggagtta gagaaggtga tgccgctggc 1260cttgccagga gcctgtctca cccagtgcat gccggagtta gagaaggtga tgccgctggc 1260
cttgcagtcc agccgcaggg acctgcctgg ctgcaccacc ccccctcctg attcgaccag 1320cttgcagtcc agccgcaggg acctgcctgg ctgcaccacc ccccctcctg attcgaccag 1320
ctgcacctgt gagtggacac cagttgcggt tgcgaccaga aacagaatga tacatgacca 1380ctgcacctgt gagtggacac cagttgcggt tgcgaccaga aacagaatga tacatgacca 1380
tcccatggaa ggtcgtctcc ttgtgggttg tggcaagctt atcatcgtgt ttttcaaagg 1440tcccatggaa ggtcgtctcc ttgtgggttg tggcaagctt atcatcgtgt ttttcaaagg 1440
aaaaccacgt ccccgtggtt cggggggcct agacgttttt ttaacctcga ctaaacacat 1500aaaaccacgt ccccgtggtt cggggggcct agacgttttt ttaacctcga ctaaacacat 1500
gtaaagcatg tgcaccgagg ccccagatca gatcccatac aatggggtac cttctgggca 1560gtaaagcatg tgcaccgagg ccccagatca gatcccatac aatggggtac cttctgggca 1560
tccttcagcc ccttgttgaa tacgcttgag gagagccatt tgactctttc cacaactatc 1620tccttcagcc ccttgttgaa tacgcttgag gagagccatt tgactctttc cacaactatc 1620
caactcacaa cgtggcactg gggttgtgcc gcctttgcag gtgtatctta tacacgtggc 1680caactcacaa cgtggcactg gggttgtgcc gcctttgcag gtgtatctta tacacgtggc 1680
ttttggccgc agaggcacct gtcgccaggt ggggggttcc gctgcctgca aagggtcgct 1740ttttggccgc agaggcacct gtcgccaggt gggggttcc gctgcctgca aagggtcgct 1740
acagacgttg tttgtcttca agaagcttcc agaggaactg cttccttcac gacattcaac 1800acagacgttg tttgtcttca agaagcttcc agaggaactg cttccttcac gacattcaac 1800
agaccttgca ttcctttggc gagaggggaa agacccctag gaatgctcgt caagaagaca 1860agaccttgca ttcctttggc gagaggggaa agaccccctag gaatgctcgt caagaagaca 1860
gggccaggtt tccgggccct cacattgcca aaagacggca atatggtgga aaataacata 1920gggccaggtt tccgggccct cacattgcca aaagacggca atatggtgga aaataacata 1920
tagacaaacg cacaccggcc ttattccaag cggcttcggc cagtaacgtt aggggggggg 1980tagacaaacg cacaccggcc ttattccaag cggcttcggc cagtaacgtt agggggggggg 1980
gagggagagg ggcgtcaaca ttctccacgg ttgaaacttt ttgtgactgg tgaggacaga 2040gagggagagg ggcgtcaaca ttctccacgg ttgaaacttt ttgtgactgg tgaggacaga 2040
ccctggtgag tgacttcaca agcatacacc ttgtgcttct cgtaatcggc cttggacagt 2100ccctggtgag tgacttcaca agcatacacc ttgtgcttct cgtaatcggc cttggacagt 2100
gtcagggtgc tggacagaga atatgtgcta tccttggagt cctgctcggt cacagactcc 2160gtcagggtgc tggacagaga atatgtgcta tccttggagt cctgctcggt cacagactcc 2160
tggctattgc cggactgcag agcgttatcc accttccact gcaccttggc ctcccgaggg 2220tggctattgc cggactgcag agcgttatcc accttccact gcaccttggc ctcccgaggg 2220
tagaaattgt tcagcaggca caccacgctg gctgtgccgg acttcagctg ctcgtcggaa 2280tagaaattgt tcagcaggca caccacgctg gctgtgccgg acttcagctg ctcgtcggaa 2280
gggggaaaga tgaacacgga gggagcggcc acggttctct tgatctccac ctttgtgccc 2340gggggaaaga tgaacacgga gggagcggcc acggttctct tgatctccac ctttgtgccc 2340
tggccaaagg tccgtggcca attagagctc tgctggcaat agtacacggc gaaatcctca 2400tggccaaagg tccgtggcca attagagctc tgctggcaat agtacacggc gaaatcctca 2400
ggctccaggg aagagattgt cagggtaaag tctgtgccag atccgcttcc ggagaaccta 2460ggctccaggg aagagattgt cagggtaaag tctgtgccag atccgcttcc ggagaaccta 2460
gcggggatgc cggtagctct gttagaggcg tcgtagatca gcagcctggg agcctggcct 2520gcggggatgc cggtagctct gttagaggcg tcgtagatca gcagcctggg agcctggcct 2520
ggcttctgct gataccaggc caggtaggag gacacggact ggctagcccg gcagctcagg 2580ggcttctgct gataccaggc caggtaggag gacacggact ggctagcccg gcagctcagg 2580
gtggccctct ctcctgggga cagagacaga gttgcggggc tctgagtcag cacaatctct 2640gtggccctct ctcctgggga cagagacaga gttgcggggc tctgagtcag cacaatctct 2640
gaatgcacac cagttgcggt tgcgaccagg aacaggatga tacaggacca tcccatagat 2700gaatgcacac cagttgcggt tgcgaccagg aacaggatga tacaggacca tcccatagat 2700
ct 2702ct 2702
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711310440.7ACN108165536A (en) | 2017-12-11 | 2017-12-11 | A kind of recombination oncolytic vaccinia virus and preparation method and application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711310440.7ACN108165536A (en) | 2017-12-11 | 2017-12-11 | A kind of recombination oncolytic vaccinia virus and preparation method and application |
| Publication Number | Publication Date |
|---|---|
| CN108165536Atrue CN108165536A (en) | 2018-06-15 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711310440.7APendingCN108165536A (en) | 2017-12-11 | 2017-12-11 | A kind of recombination oncolytic vaccinia virus and preparation method and application |
| Country | Link |
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| CN (1) | CN108165536A (en) |
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20180615 | |
| RJ01 | Rejection of invention patent application after publication |