【作者】 徐高原；

【导师】 陈焕春； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 乙型脑炎(Japanese encephalitis,JE)和伪狂犬病(Pseudorabies)均是危害养猪业的重大疫病,主要引起种猪繁殖障碍,给养猪业造成了巨大的经济损失。同时乙型脑炎还是人类中枢神经系统最常见的虫媒病之一,对人类危害巨大。猪是乙型脑炎病毒的主要扩增宿主和传染源,因此控制猪的乙型脑炎不仅对养猪业而且对人类健康都具有十分重要的意义。目前免疫接种仍是预防和控制这两种传染病最有效的措施,而现有乙型脑炎疫苗,无论是灭活疫苗还是弱毒活疫苗都存在一定的缺陷。鉴于此,本研究从乙型脑炎病毒SA14-14-2疫苗株中扩增了其主要免疫原性基因E和E-NS1;实现了E基因在大肠杆菌和哺乳动物细胞中的高效表达;构建了表达乙型脑炎病毒主要免疫原性基因E或NS1的重组伪狂犬病病毒,并对所构建的重组伪狂犬病病毒的生物学特性、安全性及免疫原进行了研究。其具体研究内容包括: 1.乙型脑炎病毒E和E-NS1基因的克隆与序列分析 根据已报道的乙型脑炎病毒SA14-14-2疫苗株的核苷酸序列,设计了2对引物,采用一步法RT-PCR扩增了其主要免疫原性基因E和E-NS1,并进行了克隆与序列测定。结果发现,与已报道的SA14-14-2疫苗株核苷酸序列比较,E基因的同源性为100%,E-NS1基因片段发生了3个核苷酸的改变,即1596 g-a、2639 t-c、2817 g-a,并导致了2个氨基酸的改变,即E207 G-R,NS114 G-S,而2639 t-c为无义突变。 2.E基因在大肠杆菌和哺乳动物细胞中的高效表达 将E基因RT-PCR产物克隆到pBlusecriptⅡSK(+)载体中,然后分别亚克隆到原核表达载体PGEX-KG和真核表达载体pcDNA3.1+中,筛选重组质粒,分别命名为pKG-E和pcDNA3.1E。pKG-E转化大肠杆菌BL21(DE3),IPTG诱导后获得高效表达,表达的融合蛋白分子量为83KD,主要以包涵体形式存在,Western blot分析证实表达产物具有良好的生物学活性。pcDNA3.1E采用脂质体法转染BHK-21细胞,48小时后收集转染的细胞处理后,进行SDS-PAGE电泳,经Western blot分析,在53KD处出现特异性反应带,而对照未出现特异性反应带,证实E基因在哺乳动物细胞中获得表达。 3.表达乙型脑炎病毒E或NSl蛋白的重组伪狂犬病病毒的构建 设计1对引物从质粒pTNSl上扩增NSl基因,双酶切后将NSl基因定向插入到伪狂犬病病毒gG缺失通用转移载体pgG-Uni中,得到重组转移载体pgG-NSl:用BamHl和EcoRI酶切pSK-E,回收E基因后与经相同酶切的载体pgG-Uni相连,得到重组转移载体pgG-E。将重组转移载体用KpnI线性化后分别与经EcoRI酶切消化的PRV Ea TK~-/gG~-/LacZ~+突变株基因组DNA共转染PK-15细胞,经空斑筛选纯化、PCR、Southern印迹、Western blot等鉴定,获得了2株纯化的重组病毒,分别命名为TK~-/gG~-/NS1~+和TK~-/gG~-/E~+。重组病毒部分生物学特性研究表明:本研究构建的2株重组病毒遗传稳定性良好:外源基因的插入不影响伪狂犬病病毒在细胞更多还原

【Abstract】 Japanese encephalitis (JE) and pseudorabies are the main causes of infectious reproductive failure in swine, and make significant economic losses in the pig industry. Also, JE is a serious mosquito-borne viral disease of major public health importance in Asia. Pigs are considered the main vertebrate host and represent an important amplifier and reservoir for JEV. Therefore prevention of JE in swine can benefit for both swine production and humans’ health. In the present, vaccination is the most effective prophylactic measure to prevent and control these diseases. However, there are problems in both inactivated and attenuated JEV vaccines that are used currently. So E and E-NS1 genes of JEV were cloned and sequenced, furthermore, E gene was expressed in E.coli and BHK-21 cell. Recombinant pseudorabies virus expressing NS1 or E protein of Japanese encephalitis virus was constructed, and the research of their biological characteristic, safety and immunogenicity was carried out.1. Cloning and sequence analysis of E and E-NS1 genes of JEV SA14-14-2E and E-NS1 genes were amplified from JEV SA14-14-2 strain by RT-PCR and were further cloned and sequenced. The results indicate that there was no nucleotide difference between the cloned E gene and E of SA14-14-2 strain, while there were 3 nucleotides and 2 amino acids differences between the cloned E-NS1 gene and E-NS1 of SA14-14-2 strain.2. Expression of E gene in E.coli and BHK-21 cellThe cloned plasmid pSK-E was digested with EcoRI and BamHI, then the fragment of E was cloned into a prokaryotic expression vector and a eucaroytic expression vector, and named pKG-E and pcDNA3.1E, respectively. The pKG-E plasmid was then transfected into E.coli BL21 (DE3). SDS-PAGE and western blotting showed that E could be highly expressed as a fusion protein whose molecular weight was about 83KD and E protein was specific to antisera against JEV. Plasmid pcDNA3.1E was transfected into BHK-21 cells with lipofectamine. After 48h, the above BHK-21 cells were harvested to analyze. Western blot indicated that E was expressed in BHK-21 cells.3. Construction of recombinant pseudorabies virus expressing NS1 or E protein of Japanese encephalitis virusE and NS1 were respectively subcloned into vector pgG-Uni to form recombinant plasmid (designed as pgG-E and pgG-NS1, respectively). A co-transfection experiment was performed in porcine kidney (PK-15) cells with pgG-E or pgG-NSl and the genome of the vector virus (PRV TK~-/gG~-/LacZ~+ mutant). By plaque purification, PCR detectionand southern hybridization, recombinant pseudorabies virus expressing E or NS1 protein oi JEV was acquired and named TK7gG"/E+ and TK7gG7NSl+, respectively. Western blot analysis and ELISA demonstrated the E or NS1 protein could be expressed in recombinant PRV strains. Studies on their biological characteristics confirmed that the propagation of recombinants in cells was not affected and their genetic stability was high. 4. Study on the immunogenicity of recombinant pseudorabies virus TK7gG7NSl+and TK/gG/E+Four to six-week-old female Balb/c mice and forty to sixty-day-old piglets that were serologically negative to both pseudorabies and Japanese encephalitis were inoculated with TK7gG7NSl+ or TKVgGTE* to evaluate their safety and immunogenicity. The results of the animal tests showed that TK7gG7NSl+ and TK7gG7E+ are safe to both mice and piglets. Furthermore, The vaccinated mice could acquire protective immunity against lethal challenge of the virulent PRV. PRV neutralizing antibody can be induced in immunized piglets, and a good humoral and cellular immune response against JEV could be induced in the mice and piglets immunized with TK7gG7E+ or TK7gG7NSl+. Although compared with JEV SA14-14-2, the specific immune responses induced by TK/gG7E+ or TK7gG7NSl+ were little lower, there was significant difference in JEV-specific CTL activity and antibody to JEV between the group immunized with TKT/gGTE* or TK/gG7NSl+ and the negative control (P>0.05). The above results revealed that the two recombinant viruses could be suitable candidate vaccine strains for developing a novel genetic vaccine to combat pseudorabies and Japanese encephalitis in the pig industry.更多还原