脊灰病毒Sabin1株疫苗载体构建及在流感通用型疫苗研制中的初步应用

发布时间：2018-07-06 20:25

本文选题：脊髓灰质炎疫苗Sabin + 1株病毒　；参考：《第四军医大学》2007年博士论文

【摘要】： 流感病毒RNA聚合酶缺少校正功能,导致基因组在复制过程中容易发生突变,人群中因以往感染产生的抗体不能有效地防止新毒株感染,因而每年需要注射流感疫苗,给社会带来沉重的经济负担。如果有一种通用型疫苗,经过全程免疫后,产生广谱性抗体,则能够预防各种变异病毒,或降低感染病毒后疾病的严重程度。甲型流感病毒囊膜上除血凝素(HA)和神经氨酸酶(NA)外,还含有一个分子量较小的基质蛋白M2,其胞外区(M2e)非常保守,是研制通用型疫苗的最佳靶位。由于M2e只含有24个氨基酸,需要以各种措施来增强其免疫原性。本论文拟将口服脊髓灰质炎疫苗(OPV)Sabin1株病毒改造为疫苗载体,采用多聚蛋白融合策略,插入流感M2e基因,将前者作为表达载体和免疫佐剂,提高人体针对M2e的免疫应答,构建一种能抵御各种甲型流感病毒的通用型疫苗。通过RT-PCR方法扩增了Sabin1株病毒基因组的三个片段f1、f2、f3并分别克隆,在f1和f2、f2和f3片段之间含重叠序列,内有基因组唯一的酶切位点。因此,三片段可以通过酶切和连接获得全基因组。将三个克隆分别测序,与GenBank中序列比较,发现在基因组7440bp中只有三个位点不同,其中26 A→G,355 C→T两个位点处于非编码区,6735 A→G导致聚合酶3D上250位氨基酸由赖氨酸变为精氨酸。说明Sabin1株基因组比较稳定,适合于作为疫苗载体。基因组起始密码子(nt743)位于pGEM-f1质粒上,通过重叠PCR方法在之前插入多克隆位点(EcoRI、XmaI、XhoI)和人工蛋白酶切位点(ALFQG)构成的功能盒(Cassette),形成pGEM-f1m质粒;同样,在位于pGEM-f2质粒上P1/P2结合处引入类似序列,构建为pGEM-f2m质粒。连接为基因组后可形成两种基本型载体PV1和PV2。多克隆位点内插入的外源基因随多聚蛋白表达后,在病毒编码的蛋白酶切割后释放外源蛋白。为提高载体RNA的转染效果,另外在载体上进行两项改造:基因组前加入锤头型核酶(Rz)基序,合成RNA后发挥核酶功能,去除多余核苷酸,产生PV基因组真正的5’-末端;在基因组后导入poly(A)40结构,后者是PV RNA感染的必要基序。两个基序均以PCR引物延伸方法产生,并通过基因置换方式导入到pGEM-f1m和pGEM-f3中,经测序证明引入的基序与设计序列一致。将改造前和改造后的质粒进行三片段连接,形成八种载体,分别含有不同的功能盒及基序组合。将携带各种PV载体的质粒线性化后,以T7 RNA聚合酶体外转录法合成RNA并纯化,通过电泳发现RNA完整性、均一性良好,适合于转染细胞。对含有Rz序列的RNA进行体外切割,经PAGE电泳检测反应进程,确定在合适的缓冲液中作用1.5小时获得最佳切割效果。采用电穿孔、脂质体转染法和磷酸钙转染法将提取的PV Sabin1 RNA转染入Vero细胞,以形成的噬斑数目比较转染效率,筛选转染大分子RNA的最佳方法。实验结果证明脂质体Tfx-20的转染效率最佳,达到1.1~1.5×103感染性克隆/μg RNA,对细胞毒性不明显。以Tfx-20转染八种载体RNA,只有同时含有poly(A) 40和Rz基序的载体pPV1RzA和pPV2RzA能有效产生活病毒,约500~800克隆/μg RNA。感染性病毒产生时间与转染RNA量有关,当以10μgRNA转染时,60小时就可以观察到细胞病变;而以1μgRNA转染时,需要4天产生病变效应。只含有poly(A)40基序的载体RNA也可以成功转染,但效率低,当RNA量为1μg时无法得到活病毒。不含两个基序或只含Rz基序的RNA在Vero细胞上无感染性,即使将RNA量增加到30μg,培养10天亦无活病毒产生。从霍乱弧菌中扩增霍乱毒素B亚单位(CTB)基因,测序证明正确后,以PCR引物延伸法构建CTB和M2e的融合基因,并在两端分别添加EcoRI和XhoI位点,通过双酶切将融合基因导入到pPV1RzA中,保持正常的编码框架,以建立的方法转染Vero细胞,获得了重组病毒rPV-CTBM2e。另外,扩增SARS-冠状病毒S蛋白的受体结合区基因S-RBD后,以同样方法获得了重组病毒rPV-S-RBD。提取重组病毒RNA,以RT-PCR方法扩增涵盖插入序列的片段并测序,证明两个重组病毒均携带预期片段。将重组病毒传代多次,通过RT-PCR方法考察重组病毒的稳定性。rPV-CTBM2e病毒传代12次后仍保持完整的外源基因,而rPV-S-RBD病毒在传代过程中丢失外源基因。rPV-CTBM2e病毒一步生长曲线证明其繁殖有延迟现象,但最终能得到和Sabin1病毒同样的滴度。将M2e合成多肽和KLH载体蛋白偶联后免疫Balb/c小鼠,制备了针对M2e多肽的单克隆抗体,以ELISA方法筛选得到7株有活性的单抗。以单抗上清检测感染Vero细胞的流感病毒,发现其中5株能与细胞表面的天然M2e结合,其中两株为强阳性,可用于重组病毒M2e表达的检测。将重组病毒感染Vero细胞,通过免疫荧光方法,分别以抗M2e单抗和抗SARS-病毒免疫血清检测,发现在细胞中出现明显的荧光信号,并集中在细胞质中,说明外源蛋白在细胞质中成功表达,与预期结果一致。本研究为研制流感通用型疫苗和SARS-CoV疫苗奠定了基础。
[Abstract]:Influenza virus RNA polymerase is lack of correction function, which causes the genome to mutate easily during the replication process. The antibody produced by the previous infection can not effectively prevent the infection of the new virus. Therefore, the influenza vaccine is injected every year to bring a heavy economic burden to the society. If there is a general type vaccine, after the whole course of immunization, The production of broad-spectrum antibodies can prevent a variety of variant viruses, or reduce the severity of the disease after infection. In addition to HA and NA, the envelope of influenza A virus also contains a small molecular weight matrix protein M2, and its extracellular domain (M2e) is very conservative. It is the best target for the development of universal vaccine. Because M2e With only 24 amino acids, it is necessary to enhance its immunogenicity with various measures. This paper intends to transform the oral poliomyelitis vaccine (OPV) Sabin1 strain virus into a vaccine carrier, and inserts the M2e gene into the influenza virus by using the fusion strategy of polyprotein, the former as an expression vector and an immune adjuvant, to improve the immune response to M2e, and to construct a kind of immune response to the human body. A universal vaccine that can resist all kinds of influenza A virus.
Three fragments of Sabin1 virus genome were amplified by RT-PCR method, F1, F2, F3 and cloned respectively. There were overlapping sequences between F1 and F2, F2 and F3 fragments, and the only enzyme cut site was in the genome. Therefore, the whole genome was obtained by the three fragment by enzyme cut and connection. The three clones were sequenced and found in the GenBank sequence. There are only three different loci in genomic 7440bp, of which 26 A, G and 355 C to T are in the non coding region. 6735 A to G causes the 250 amino acids on the polymerase 3D to arginine. It shows that the genome of the Sabin1 strain is more stable and is suitable for the vaccine carrier.
The genomic initiation codon (nt743) is located on the pGEM-f1 plasmid. The function box (Cassette), which is composed of EcoRI, XmaI, XhoI, and ALFQG, is inserted before the PCR method to form a pGEM-f1m plasmid. Similarly, a similar sequence is introduced into the P1/P2 binding site on the pGEM-f2 plasmid, which is constructed as a pGEM-f2m plasmid. After being inserted into the genome, the exogenous genes inserted in the polyclonal loci of the two basic carriers, PV1 and PV2., were expressed with the polyproteins, and the exogenous proteins were released after the virus encoded protease was cut.
In order to improve the transfection effect of carrier RNA, two modifications were carried out on the carrier: before the genome was added to the hammer head ribozyme (Rz), the function of the ribozyme was synthesized and the redundant nucleotides were removed and the true 5 '- terminal of the PV genome was produced; the poly (A) 40 structure was introduced after the genome, and the latter was the necessary order of PV RNA infection. Two sequences were all P The CR primer extension method was produced and introduced into pGEM-f1m and pGEM-f3 by gene replacement. The sequence was sequenced to agree with the design sequence. The modified and reformed plasmids were connected by three fragments to form eight carriers and contain different functional boxes and sequence combinations respectively.
After linearizing the plasmids carrying various PV carriers, RNA was synthesized by T7 RNA polymerase in vitro transcription. The integrity of RNA was found by electrophoresis, and the homogeneity was good. It was suitable for transfection of cells. The RNA in Rz sequence was cut in vitro and the reaction process was detected by PAGE electrophoresis. It was determined that the best effect was obtained in the appropriate buffer solution for 1.5 hours. Cutting effect.
The extracted PV Sabin1 RNA was transfected into Vero cells by electroporation, liposome transfection and calcium phosphate transfection. The best method of transfection of large molecule RNA was obtained by comparing the number of plaques formed by the number of plaque. The results showed that the transfection efficiency of liposome Tfx-20 was the best, reaching 1.1 ~1.5 x 103 infectious clones / micronux RNA. It is obvious that the transfection of eight carrier RNA with Tfx-20, only the carrier pPV1RzA and pPV2RzA containing poly (A) 40 and Rz motif, can produce a living virus effectively, and the time for the production of 500~800 cloned / Muu RNA. infective virus is related to the RNA quantity of transfection. When the transfection is 10 Mu gRNA, the cell lesion can be observed in 60 hours; and it takes 4 days when the transfection is 1 mu. The vector RNA that contains only poly (A) 40 can also be transfected successfully, but the efficiency is low. When the amount of RNA is 1 mu g, the live virus can not be obtained. No two motif or only Rz based RNA is not infected on Vero cells. Even if RNA is increased to 30 mu G, no live virus is produced for 10 days.
The gene of cholera toxin B subunit (CTB) was amplified from Vibrio cholerae. After the sequencing proved to be correct, the fusion gene of CTB and M2e was constructed by PCR primer extension method, and EcoRI and XhoI loci were added at both ends respectively. The fusion gene was introduced into pPV1RzA by double enzyme cutting, and the normal coding frame was maintained. The transfection of Vero cells was obtained by the method of establishment. Recombinant virus rPV-CTBM2e., after amplification of the receptor binding region gene S-RBD of the SARS- coronavirus S protein, obtained the recombinant virus rPV-S-RBD. from the recombinant virus rPV-S-RBD. to extract the recombinant virus RNA, and amplified the inserted sequence by RT-PCR method and sequenced it. It was proved that all the two recombinant viruses carried the expected fragment.
The recombinant virus was passed on several times, and the RT-PCR method was used to investigate the stability of the recombinant virus.RPV-CTBM2e virus for 12 times, and the whole foreign gene remained intact after 12 times, while the one step growth curve of the exogenous gene.RPV-CTBM2e virus was lost during the passage of the virus, which proved that the propagation of the recombinant virus was delayed, but eventually it could be similar to that of the Sabin1 virus. The titer.
After coupling M2e synthetic polypeptide and KLH carrier protein to immunize Balb/c mice, a monoclonal antibody against M2e polypeptide was prepared, and 7 active McAbs were screened by ELISA method. The detection of influenza virus in Vero cells was detected by the monoclonal antibody supernatant. It was found that 5 of them were capable of combining with the cell surface of Tian ran M2e, of which two were strongly positive and could be used for heavy weight. Detection of M2e expression in group virus.
The recombinant virus infected Vero cells was detected by immunofluorescence method and detected by anti M2e monoclonal antibody and anti SARS- virus sera respectively. It was found that the obvious fluorescent signal was found in the cell and concentrated in the cytoplasm, indicating that the exogenous protein was successfully expressed in the cytoplasm, which was the same as expected fruit. This study is to develop a general influenza vaccine and SARS. The -CoV vaccine lays the foundation.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【相似文献】