新型JEV复制子载体的构建及表达炭疽PA的JEV复制型DNA载体的免疫原性研究

发布时间：2018-06-18 05:33

本文选题：日本脑炎病毒 + 复制子　；参考：《中国人民解放军军事医学科学院》2009年硕士论文

【摘要】： 黄病毒属包括70多种病毒,其中绝大多数是节肢动物传播的人类病原。日本脑炎(JE)是一种蚊虫传播的病毒疾病,年发病率约为十万分之一,病死率约为5-40%,另有20-40%会引起严重的神经性疾病和后遗症。我国除新疆,西藏,青海外均有发病,现在每年仍有几千例病人,对人民的健康造成极大的危害,被卫生部列为乙类传染病。黄病毒基因组有一个显著特性,其RNA具有自主复制能力、同时具有感染性,裸RNA进入敏感细胞后,能够大量自主复制,并且进一步翻译成相应的病毒蛋白,在胞内包装成为完整的病毒颗粒。黄病毒复制子通常指将基因组上的结构基因部分切割下来,而保留完整的非结构蛋白基因,这样亚基因组RNA保留了自主复制能力,其不但能够表达非结构蛋白,而且能够表达保留的结构蛋白和/或添加的外源基因。理论上讲,这种亚基因组的复制能力等同于完整的基因组,正链RNA呈指数级增加。如果在这种复制子中插入外源基因,由于复制子RNA在细胞胞浆内大量扩增,就使得外源基因得到有效表达,因此,复制子RNA可作为表达外源基因的良好载体。目前,黄病毒中的KUN、DEN和YFV复制子已经被报道可以作为发展新型疫苗的潜在的有用的工具。而在本研究中,我们希望以JEV SA14-14-2株为基础,构建JEV亚基因组复制子,并进一步建立JEV复制子载体系统,用于更多的疫苗研究。我们先后构建了pMW-G2R~pMW-G4R系列JEV复制子。这些复制子的主要区别在于结构蛋白编码序列删除的方式不同,MCS,FMDV-2A等序列的添加。复制子的稳定性是构建复制子的一个主要问题,我们先后选用了不同的质粒载体用于作为复制子的骨架载体,为了促进复制子的稳定性,最终我们使用了一个极低拷贝但稳定性很好的细菌质粒载体pMW-118。另外,我们为了后续实验操作的方便,以及提高复制子载体的表达效力,在复制子载体上添加了CMV启动子序列,构建了pCMW-2M复制子载体,多次测序证明JEV复制子pCMW-2M于30℃在大肠杆菌Top10繁殖过程中十分稳定,没有突变、插入或缺失。我们通过间接免疫荧光实验(IFA)分析了JEV复制子转染BHK-21细胞病毒蛋白的表达。抗原主要定位在胞浆。这些抗原阳性的细胞同对照细胞形状、大小一致。在JEV抗原阳性的细胞没有观察到任何明显的细胞病变效应(CPE)。pMW-G2R~pMW-G4R和pCMW-2M复制子转染细胞IFA的阳性率差别很大,从1%~30%不等,表明C蛋白的完整性、CMV启动子的存在及非结构蛋白基因的正确表达对复制子载体表达病毒蛋白的能力影响至关重要。我们分别将绿色荧光蛋白(EGFP)和荧光素酶(Luc)报告基因插入复制子载体pCMW-2M。p2MEGFP转染BHK-21细胞24小时后开始观察EGFP的表达。阳性细胞的形状、大小同对照BHK-21细胞没有差别,同时也没有观察到细胞病变效应。EGFP阳性细胞在转染后24小时很少,在转染后72小时明显。与观察EGFP表达类似,荧光素酶基因的表达在转染后7天仍然能够检测到,荧光素酶信号在转染后24小时到96小时呈指数增长。这些结果表明了插入了外源基因的JEV复制子能够有效地表达外源基因。我们通过免疫小鼠来研究JEV复制子pCMW-2M和pCMW-G2R的免疫原性。按每只小鼠50μg质粒pCMW-2M剂量肌肉注射6周龄的雌BALB/c小鼠,3次免疫后诱导的抗JEV的抗体滴度为1:1270、中和抗体滴度为1:3(250%噬斑减少实验),略高于pCMW-G2R组。pCMW-2M组的T淋巴细胞刺激指数(SI)为2.8,略高于pCMW-G2R组。这些结果证明了构建的JEV复制子载体pCMW-2M及pCMW-G2R在小鼠体内可以诱导针对JEV的体液免疫和细胞免疫。另外,pCMW-2M及pCMW-G2R免疫小鼠血清对JEV病毒攻击具有较好的保护作用,可达到约70%存活。炭疽杆菌(Bacillus anthracis)是一种能形成芽胞的革兰氏阳性菌,由它感染引起的炭疽病是严重危害人类的一种致死性的重大传染病,同时它又是一种潜在的生物战剂和生物恐怖试剂。PA是当前应用的疫苗主要成分,是引起机体免疫应答的最有效的免疫原。为了发展安全、有效的炭疽疫苗,DNA疫苗是一个重要方向。我们将PA抗原的第四结构域基因插入到JEV复制子pCMW-2M,这个插入了PA4基因的JEV复制子以DNA形式免疫小鼠后诱导了良好的体液免疫和细胞免疫反应,免疫3次后抗体滴度达到1:36200,T淋巴细胞刺激指数为2.6,这是国内外首次对构建表达炭疽PA的JEV复制型DNA载体进行的初步探索。另外,我们还表达并纯化了黄热病毒YFV囊膜蛋白(E蛋白)结构域Ⅲ,研究其作为亚单位疫苗预防YFV、JEV感染的可能性。YFDⅢ蛋白在大肠杆菌高效可溶性表达,表达量约占菌体蛋白的50%。纯化的YFDⅢ蛋白免疫新西兰兔和BALB/C鼠。Western Blot分析及ELISA表明纯化后的表达产物具有良好的抗原性和免疫原性。利用纯化的YFDⅢ蛋白免疫新西兰兔,获得了高达1:4×105滴度的抗YFV抗体和1:2×104滴度的抗JEV抗体;利用纯化的YFDⅢ蛋白免疫BALB/C鼠,获得了1:7×104滴度的抗YFV抗体和1:2×103滴度的抗JEV抗体。表明能产生对抗YFV和JEV的抗体,抗原性及免疫原性较好。同时抗YFV免疫血清对乳鼠YFV攻毒的保护作用实验结果表明YFDⅢ蛋白免疫小鼠血清对YFV病毒攻击有较好的保护作用,这些结果提示YFDⅢ蛋白有可能具有开发研制成亚单位疫苗的潜能,这是国内外首次利用大肠杆菌高效可溶性表达了YFDⅢ蛋白并证明其具有较好的免疫原性和保护效力。综上所述,我们成功构建了新型JEV复制子载体,进行了国内外首次基于JEV减毒活疫苗株SA14-14-2的复制子载体的构建、表达自身和外源蛋白能力、免疫原性等方面的系统研究,证明JEV复制子具有作为递送外源基因的良好载体的潜力,为研究JEV的结构蛋白与非结构蛋白在病毒复制、包装中的作用积累了重要资料,为进一步研究JEV非结构蛋白(主要是NS1蛋白)的免疫原性及保护效力提供了帮助,为研制新型表达炭疽PA的JEV复制型DNA载体甚至双价或多价复制型DNA载体奠定了基础,同时也为利用JEV复制子载体作为疫苗载体进一步研制其它新型DNA疫苗建立了一个有价值的技术平台。
[Abstract]:The genus yellows includes more than 70 viruses, most of which are the human pathogens that are transmitted by arthropods. Japanese encephalitis (JE) is a mosquito borne virus disease with an annual incidence of about 1/100000, the mortality rate is about 5-40%, and 20-40% can cause severe neurological diseases and sequelae in China, except in Xinjiang, Tibet, and Qinghai, Every year, thousands of patients are still suffering great harm to the health of the people. They are classified as class B infectious diseases by the Ministry of health.
The genome of the yellow virus has a significant characteristic. The RNA has the ability to replicate autonomously, and has the infection. After the naked RNA enters the sensitive cell, it can be replicated in a large amount, and further translated into the corresponding virus protein and packaged into a complete virus particle in the cell. The yellow disease virus replicon usually refers to the structural gene part on the genome. The subgenome RNA preserves the intact non structural protein gene so that the subgenomic DNA preserves the independent replication ability, which not only expresses the unstructured protein, but also expresses the retained structural protein and / or the added exogenous gene. In theory, the subgenome is reproduced with the complete genome, and the positive chain RNA is exponentially. If the extraneous gene is inserted in this replicon, the extraneous gene is effectively expressed because the replicon RNA is amplified in the cytoplasm. Therefore, the replicon RNA can be used as a good carrier for the expression of foreign genes. At present, the KUN, DEN and YFV replicas in the yellow virus have been reported to be used as a new type of vaccine. Potential useful tools. In this study, we hope to build the JEV subgenome replicon on the basis of the JEV SA14-14-2 strain and further establish a JEV replicator system for more vaccine research.
We have successively constructed the pMW-G2R~pMW-G4R series JEV replicon. The main difference between these replicas lies in the different ways of deletion of the structural protein coding sequence, the addition of MCS, FMDV-2A and other sequences. The stability of the replicon is a major problem for the construction of the replicon. We have selected the different plasmid vectors for the replicon bone. In order to promote the stability of the replicator, in order to promote the stability of the replicator, we finally used a very low copy but stable bacterial plasmid carrier pMW-118.. We added the CMV promoter sequence on the replicator for the convenience of the follow-up experiments and the enhancement of the expression effectiveness of the replicator carrier, and constructed the pCMW-2M replication subcarrier. Repeated sequencing showed that the JEV replicon pCMW-2M was stable at 30 C at the temperature of Escherichia coli Top10, without mutation, insertion or deletion.
We analyzed the expression of BHK-21 cell virus protein transfected by JEV replicator by indirect immunofluorescence assay (IFA). The antigen was mainly located in the cytoplasm. These antigen positive cells were in the same shape as the control cells. No obvious cytopathic effect (CPE).PMW-G2R~pMW-G4R and pCMW-2M were observed in the JEV antigen positive cells. The positive rate of the replicon transfected cell IFA is very different, which is different from 1%~30%, indicating the integrity of the C protein, the existence of the CMV promoter and the correct expression of the non structural protein gene, which are very important to the ability of the replicator to express the virus protein.
We observed the expression of EGFP by inserting the green fluorescent protein (EGFP) and luciferase (Luc) reporter gene into the replicator pCMW-2M.p2MEGFP and transfecting BHK-21 cells for 24 hours. The shape and size of the positive cells were not different from those of the control BHK-21 cells. At the same time, there was no observation that the cell lesion effect of.EGFP positive cells was 24 after transfection. There were few hours, 72 hours after transfection. Similar to the expression of EGFP, the expression of luciferase gene could still be detected at 7 days after transfection, and the luciferase signal increased exponentially at 24 hours to 96 hours after transfection. These results showed that the JEV replica inserted into the foreign gene could effectively express the foreign gene.
We studied the immunogenicity of JEV replicator pCMW-2M and pCMW-G2R by immunizing mice. By intramuscular injection of 6 weeks old female BALB/c mice at the dose of 50 mu g plasmid per mouse, the antibody titer of anti JEV induced by 3 immunity was 1:1270, and the neutralizing antibody titer was 1:3 (250% plaque reduction test), slightly higher than the T lymph nodes in the.PCMW-2M group of the pCMW-G2R group. The cell stimulation index (SI) was 2.8, slightly higher than that in the pCMW-G2R group. These results showed that the constructed JEV replicator, pCMW-2M and pCMW-G2R, could induce humoral and cellular immunity against JEV in mice. In addition, the serum of pCMW-2M and pCMW-G2R immune mice had a good protective effect on the JEV virus attack, and could reach about 70%. Bacillus anthracis is a gram positive bacterium that can form a bud. Anthracnose caused by it is a fatal major infectious disease that seriously endangers human beings. At the same time, it is a potential biological warfare agent and bioterrorism reagent.PA, the main component of the current vaccine, and the most important immune response to the body. Effective immunogen. In order to develop a safe and effective anthrax vaccine, DNA vaccine is an important direction. We insert the fourth domain gene of PA antigen into the JEV replicon pCMW-2M. This JEV replicator, inserted in the PA4 gene, immunized mice in the form of DNA and induced a good body liquid immunity and cellular immune response, and the antibody was immunized for 3 times. Titer reached 1:36200 and T lymphocyte stimulation index was 2.6. This is the first exploration of constructing JEV replication DNA vector expressing anthrax PA for the first time at home and abroad.
In addition, we also expressed and purified the YFV capsule protein (E protein) domain of the yellow fever virus (HDV) III, and studied the possibility of its subunit vaccine as a vaccine to prevent YFV, the possibility of JEV infection, the expression of.YFD III protein in Escherichia coli, and the expression of the YFD III protein of the 50%. purification of the bacterial protein, the.Western Blot analysis of New Zealand rabbits and BALB/C mice. And ELISA showed that the purified expression product had good antigenicity and immunogenicity. The purified YFD III protein was used to immunize New Zealand rabbits to obtain anti YFV antibody and 1:2 x 104 titer with a 1:4 x 105 titer, and the purified YFD III protein was used to immunize BALB/C mice, and the anti YFV antibody and 1:2 x 103 titer of 1:7 x 104 titre were obtained. Anti JEV antibody. It showed that antibodies against YFV and JEV were produced, antigenicity and immunogenicity were better. Meanwhile, the protective effect of anti YFV immunized serum against YFV in milk mice showed that YFD III protein immunized with mice serum had a good protective effect on the attack of YFV virus. These results suggest that YFD III protein may have development and development. The potential of the unit vaccine is the first use of Escherichia coli to efficiently express YFD III protein and prove that it has good immunogenicity and protective effect.
To sum up, we successfully constructed a new JEV replicator, and carried out the construction of the first replicating carrier based on the JEV live attenuated vaccine strain SA14-14-2, expressing the ability of self and exogenous protein, immunogenicity and so on. It proved that the JEV replicator has the potential as a good carrier for delivery of foreign genes. The role of JEV's structural proteins and unstructured proteins in the replication and packaging of the virus has accumulated important information, which helps to further study the immunogenicity and protective effect of the JEV non structural protein (mainly NS1 protein), and provides a new JEV replicative DNA carrier for the expression of anthrax PA and even the bivalent or multivalent replicative DNA carrier. It also provides a valuable technology platform for further development of other DNA vaccines by using JEV replicon vectors as vaccine vectors.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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