【作者】 徐静；

【导师】 郭万柱；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2008， 博士

【摘要】 乙型脑炎病毒和伪狂犬病病毒都是引起母猪繁殖障碍的重要病原之一,给养猪业造成巨大的经济损失。而且乙型脑炎是一种人畜共患病,猪是乙脑病毒的主要放大宿主,因此对猪乙脑病毒的控制具有十分重要的公共卫生意义。两种病原都是病毒,预防控制的主要措施除了常规的生物安全外,就是疫苗预防接种。目前尚无家畜专用的JEV疫苗株,主要用人类疫苗使用的弱毒株和强毒株。同时由于现代化养殖中猪群频繁接种,对猪群造成很大的应激,严重影响生产性能。目前,国际上动物用疫苗的发展趋势是标记疫苗及其配套的监测方法。鉴于此,本研究从乙脑病毒CQRC-1株扩增了主要免疫原性基因E、PrM,构建了两株表达乙脑病毒主要免疫原性基因的重组伪狂犬病病毒,即SA215（E）和SA215（F）,并对两株重组病毒进行生物学特性研究。同时重组疫苗接种后需要一种特异的血清学鉴别方法,鉴于此对乙脑病毒非结构蛋白基因NS1进行原核表达,并建立了ELSIA方法。其主要的研究内容如下:1.乙脑病毒PrM、E、NS1和NS3基因的克隆和测序根据已报道的乙脑病毒CQRC-1株的核苷酸序列设计四对引物,在上下游引物的5’端引入一酶切位点。采用RT-PCR方法,扩增了PrM、E、NS1和NS3基因,分别利用TA克隆插入pMD18-Tsimple载体,命名为PrM-T、E-T、NS1-T和NS3-T。测序结果表明E基因与已公布的SA14、JaGAr01和P3株核苷酸同源性分别为97、97、96%;PrM同源性分别为97、97、96%;NS1同源性高达99、98、98%;NS3核苷酸同源性分别为99、98、98%;与报道的CQRC-1的对应序列相同。2.表达JEV E和PrM基因的重组伪狂犬病病毒的构建从E-T和PrM-T酶切获得JEV的E基因和PrM基因,分别插入到PRV gI基因缺失通用转移载体pPI-2.EGFP的多克隆位点中,构建转移质粒,命名为PPE和PPM。然后将质粒PPE和PPM分别与PRV基因缺失株SA215基因组共转染Vero细胞,通过同源重组,构建了融合表达JEV E基因和JEV PrM基因的重组伪狂犬病毒株,命名为SA215（E）和SA215（F）。经荧光检测、PCR、Southern转印杂交鉴定,结果表明SA215（E）和SA215（F）构建成功;Western免疫印迹检测表明JEV E基因和JEV PrM基因在重组病毒内获得表达,SA215（E）产生大小约90kD的融合蛋白;而SA215（F）产生大小约60kD融合蛋白,并且,表明产生的融合蛋白具有反应原性。3.重组病毒SA215（E）、（F）部分生物学特性及稳定性SA215（E）、（F）和SA215对Vero细胞的致病变效应、噬斑形态、一步法生长曲线试验以及电子显微镜观察病毒粒子形态特征。结果表明SA215在插入了JEV E或PrM基因后,并不改变其生物学特性。SA215（E）、（F）连续传6带,在荧光显微镜下可见荧光,用PCR跟踪检测仍可扩增出JEV E基因或PrM基因,表明SA215（E）、（F）至少在6代内稳定。4.PRV-JEV二联基因工程疫苗对仔猪、小鼠和家兔的免疫原性和安全性试验将重组伪狂犬病毒SA215（E）和（F）制成PRV-JEV二联基因工程疫苗接种小鼠、家兔和仔猪。并对该疫苗的免疫原性和安全性进行评价。重组伪狂犬病毒SA215（E）、SA215（F）、SA215和野毒Fa株分别以10⁵TCID₅₀剂量接种小鼠,结果接种Fa株小鼠全部死亡,而接种SA215（E）、（F）和SA215株的小鼠全部存活;将四株分别以10⁷TCID₅₀接种家兔,接种Fa的家兔在14天全都死亡,而接种SA215（E）、（F）和SA215株的家兔全部存活;初生仔猪接种SA215（E）和SA215（F）后除前三天体温升高外,其他正常。这些结果表明SA215（E）和（F）株的毒力显著低于PRV野毒株,而与亲本株SA215相近,初步验证了重组病毒的安全性。将PRV-JEV二联基因工程疫苗SA215（E）、（F）及SA215分别加强免疫小鼠后,腹膜内攻毒JEV强毒株CQRC-1,结果显示SA215（E）能100%（8/8）的保护,SA215（F）仅50%（4/8）保护,而SA215完全不能保护小鼠JEV攻毒（0/8）;血清中和试验表明SA215（E）接种两次后即首免后28天中和抗体滴度达1:20,而SA215（F）仅为1:4。SA215（E）、SA215（F）和SA215以10⁷TCID₅₀接种家兔两次后,用10⁷TCID₅₀ PRV Fa株攻毒,SA215（E）、（F）和SA215都能100%（4/4）保护PRV强毒攻毒。淋巴细胞增殖试验（MTT法）结果显示SA215（E）和（F）能诱导小鼠特异脾淋巴细胞的增殖;而T细胞亚类数量的检测结果显示SA215（E）和SA215（F）免疫小鼠能有效提高T细胞数量,增强小鼠的免疫水平。这些结果表明重组伪狂犬病毒SA215（E）和SA215（F）制成的PRV-JEV二联基因工程疫苗能成功诱导小鼠产生细胞免疫和体液免疫应答。以上结果表明重组病毒SA215（E）可以作为预防猪乙脑和伪狂犬病的二联基因工程标记疫苗候选株。5.对已构建好的重组质粒NS1-T和NS3-T通过双酶切后。得到JEV NS1和NS3基因,将其亚克隆入原核表达载体pET-32a（+）中。在IPTG的诱导下,NS1和NS3基因以融合蛋白的形式进行表达。通过表达条件的优化,确立了NS1蛋白表达时的最佳诱导物浓度为0.4 mmol/L,诱导时间为4 h,诱导温度为37℃。以纯化的融合蛋白为抗原包被酶标板,优化抗原最佳包被浓度为10μg/ml,血清稀释度1:40,最适封闭液为1%BSA,血清反应时间为90min,酶标二抗HRP-SPA的工作浓度为1:2000,反应时间为60min,底物在室温显色时间为10min。根据已建立的ELISA方法及反应条件,确定阴阳性临界值为0.152。以建立的方法检测JEV血清学反应,能区分活毒感染与灭活疫苗接种,可作为二联基因工程疫苗SA215（E）的配套监测方法。更多还原

【Abstract】 Japanese Encephalitis (JE) and Pseudorabies are the main causes of infectious reproductive failure in swine, and make significant economic losses in swine industry. In addition, JE is a serious mosquito-borne viral disease of major Public health importance in Asia and Australia.Pigs are considered the main vertebrate host and represent an important amplifier and reservoir for JEV. Therefore prevention and control of JE in swine can benefit for both swine production and ’human being’ public health. At present, besides biosecurity measures, vaccination is the most effective measure to prevent and control these diseases. However, there is no JEV strain targeted specially to swine vaccination, so there are problems in both inactivated and attenuated JEV vaccines used currently. In addition, the stresses caused by administrating vaccination frequently deteriorate the performance of pigs. In practice, multivalent vaccines are preferred. For the development of a bi-linked vaccine, So, E and PrM genes of JEV were amplified, cloned and sequenced. The recombinant pseudorabies viruses were constructed and the biological characteristics were evaluated. To differentiate infections of wild JEV and the recombinant viruses, an ELISA was developed with the NS1 protein expressed in prokaryotic cells as coating antigen.1. Cloning and sequencing of PrM, E, NS1 and NS3 genes of JEVFour pairs of primers were synthesized based on the nucleotide acid sequence of JEV CQRC-1 strain, and the appropriate restriction enzyme sites were introduced into the primers. PrM, E, NS1 and NS3 genes of JEV were generated by RT-PCR, then were cloned into pMD18-T simple vector, and designated PrM-T, E-T, NS1-T and NS3-T. PrM, E, NS1 and NS3 of strain CQRC-1 shared 96-97%, 96-97%, 98-99% and 98-99% nucleotide identities with strains SA14、JaGAr01 and P3, respectively.2. Construction of recombinant pseudorabies viruses expressing PrM or E protein of Japanese encephalitis virusThe transfer plasmids pPI-2.EGFP.PrM and pPI-2.EGFP.E were constructed by inserting the PrM and E gene of JEV into the multiple clone sites of universal transfer vector pPI-2.EGFP, respectively. The recombinant pseudorabies viruses were rescued by co-transfecting the pPI-2.EGFP.PrM and pPI-2.EGFP.E with the genome of SA215, respectively. The recombinant pseudorabies viruses were identified by fluorescence demonstration, PCR test and southern blotting, then named SA215 (E) and SA215 (F). The result of western blot analysis demonstrated the E and PrM protein could be expressed in recombinant PRV strains. The results showed that the recombinant pseudorabies viruses were successfully constructed.3. Biological characteristics and genetic stability of the recombinant viruses SA215 (E) and SA215 (F)Comparison of cytopathogenic effect, plaque form and one step growth curve in Vero cell and the virion characteristics between SA215(E), SA215(F) and SA215 confirmed no effect on SA215 biological characteristics after inserting PrM or E gene into genome of SA215. After six successive passages in Vero cell, the observation of fluorescence and PCR detection results indicated that E and PrM genes had been stably inserted into the genome of the recombinant viruses, and that the recombinant viruses SA215(E) and SA215(F) genetic stability were high.4. Evaluation of the immunogenicity and safety of two bi-linked genetic engineering vaccinesTo evaluate the safety and immunogenicity of the two bi-linked genetic engineering vaccines, Balb/c mice aged 6 weeks, piglets aged 1 day and rabbits weighted 1.5kg that were serologically negative to both pseudorabies virus and Japanese encephalitis virus were selected. The results confirmed that both of SA215 (E) and SA215 (F) are safe to mice, rabbits and piglets. All mice challenged with lethal dose of virulent JEV were protected by SA215(E), by contrast, only half of the mice were protected by SA215(F).The rabbits vaccinated with them could acquire protect immune against lethal dose challenge of the virulent PRV strain Fa. Furthermore, the specific immunological responses could be induced in vaccinated animals. SA215 (E) and SA215 (F) elicited neutralization antibody to JEV and JEV-specific CTL activity. But those of SA215 (E) were higher than that of SA215 (F). Neutralization antibody titer in the mice vaccinated two doses with SA215 (E) or SA215 (F) reached 1:20 and 1:4, respectively.The above results revealed that the recombinant strain SA215 (E) could be potentially used as bi-linked marker vaccine against JEV and PRV in swine industry.5. Development of indirect ELISA with NS1The recombinant plasmids NS1-T and NS3-T were digested with BamH I/HindⅢand KpnI/EcoRI to generate NS1 and NS3 genes, and subcloned into the prokaryotic expression vector pET-32a(+), respectively. The recombinant plasmids were transformed into E.coli BL21. The transferred bacteria were induced by IPTG. The expression of NS1 protein was optimized with proper inducing conditions of 0.4 mmol/L IPTG, 4 hours and 37℃. Using the purified fusion protein as coating antigen, the optimal concentration of NS1 for coating of plate was 10μg/ml; the dilution of sera was 1:40; the best blocking solution was 1% BSA; serum sample for detection should be incubated for 90min; the working concentration of HRP-SPA was 1:2000; HRP-SPA should be incubated for 60min at 37℃; the substrate for ELISA was incubated at RT for 10min. Following the determination of condition of ELISA, the threshold value of ELISA was 0.152. Serological results by the indirect enzyme-linked immunosorbent assay (ELISA) indicated that the assay could differentiate infection with vaccination, and could be used as accompanied method to the surveillance of the SA215 (E) in this experiment.更多还原