日本脑炎病毒WHe株基因组特征及其疫苗的研究

发布时间：2018-04-25 07:20

本文选题：日本脑炎病毒 + WHe株　；参考：《西北农林科技大学》2007年硕士论文

【摘要】： 日本脑炎(Japanese Encephalitis,JE),又称流行性乙型脑炎,是由日本脑炎病毒(Japanese Encephalitis Virus,JEV)引起的一种中枢神经系统人畜共患急性传染病。其致死率高达30%左右,50%的患者会留下永久性的后遗症,其中,中国的JE发病数占世界总发病数的80%以上。库蚊伊蚊是该病的主要传播媒介,而猪则是重要的传染源和放大器。猪患JE的主要表现为怀孕母猪流产、产死胎、弱仔、公猪睾丸炎及少数仔猪呈神经症状,给养猪业造成了巨大的经济损失。目前,用于JE免疫预防的疫苗有灭活疫苗和弱毒疫苗两种,其安全性和较高的生产应用成本已成为人们日益关注的问题。近年来,病毒分子生物学和反向遗传学的发展为开发新型JE疫苗提供了新的技术手段。本研究为进一步探索JEV/WHe株基因组功能、致病机理及构建高效新型疫苗,开展了以下工作: 1.JEV/WHe株全基因组特征分析本研究设计了11对特异性引物,采用RT-PCR技术扩增得到覆盖JEV/WHe株基因组全长cDNA的11个特异性片段,测序分析表明,JEV/WHe株基因组由一个开放阅读框(ORF,10296个核苷酸)和5’端非编码区(5’LITR,95个核苷酸)和3’端非编码区(3’UTR,585个核苷酸)组成。核苷酸序列和氨基酸序列分析表明,WHe株与P3株亲缘关系最近,但3’UTR.形成的二级结构与Vellore P20778株3’UTR.形成的二级结构最相似;与黄病毒科其他属成员比较,3’UTR最末端约84个核苷酸所形成的二级结构相当保守。 WHe株与P3株的膜蛋白(E)拥有M(76)、G(306)和L(408)三个特有氨基酸,而在其它19株JEV为T(76)、E(306)、S(408),推测可能与病毒的宿主及组织嗜性有关。WHe株E蛋白中存在控制黄病毒毒力的RGD(387-389)基序,而一部分弱毒株也有该基序,提示JVE毒力型的决定簇具有毒株特异性。由全基因组序列构建的系统发育图与由E基因构建的系统发育图仅有一些细微的差别,表明E基因在研究各分离株之间进化关系中具有重要意义;从GenBank中调出不同时间和地区分离的140株JEV E基因,构建了系统发育图谱。 2.JEV/WHe株全长cDNA的长链RT-PCR法扩增采用长链RT-PCR技术扩增基因组全长eDNA。对其进行PCR扩增鉴定,并对含复杂二级结构的3’末端和5’末端非编码区扩增片段进行了测序分析。扩增结果表明,扩增出的JEV WHe株基因组全长cDNA分子近11kb大小:非编码区测序分析表明,扩增产物为WHe株所特有。证明长链RT-PCR法可用于JEV W-He株基因组全长cDNA的扩增。 3.JEV/WHe株DNA疫苗载体构建采用RT-PCR.技术扩增prM/E和NSl全基因并进行序列分析,将其分别克隆于真核表达载体pAVXI.,构建的表达载体pAVXlprM/E和pAVXl-NSl,酶切鉴定和序列分析表明,JEV/WHe株DNA疫苗载体构建成功。 4.WIte株NSl基因在大肠杆菌中的表达. 将NSl基因克隆到pET28b(+)表达载体,构建了pET28b(+).NSl重组表达载体,转化表达宿主大肠杆菌BL21(DE3),并对其进行了诱导表达,对表达产物进行检测。结果表明,表达产物相对分子质量约为43ku。为JE亚单位疫苗和DNA疫苗的开发奠定了基础。
[Abstract]:Japanese encephalitis (Japanese Encephalitis, JE), also known as epidemic encephalitis, is an acute infectious disease of the central nervous system caused by Japanese encephalitis virus (Japanese Encephalitis Virus, JEV). Its mortality rate is up to 30%, and 50% of the patients will leave a permanent sequelae, of which the number of JE in China accounts for the world's total hair. The number of diseases is more than 80%. Aedes Culex is the main vector of the disease, and the pig is an important source of infection and amplifier. The main performance of JE is pregnancy sow abortion, stillbirth, weak piglet, boar orchitis and a few piglets with neurologic symptoms, which have caused huge economic loss to the pig industry. At present, vaccines for JE immunization are extinguishing. In recent years, the development of molecular biology and reverse genetics has provided a new technical means for the development of new JE vaccines.
In order to further explore the genome function, pathogenic mechanism and construction of efficient new vaccine of JEV/WHe strain, we carried out the following work:
Genomic characteristics analysis of 1.JEV/WHe strain
In this study, 11 pairs of specific primers were designed, and 11 specific fragments were amplified by RT-PCR technology to cover the total length of the genome of JEV/WHe strains. The sequence analysis showed that the JEV/WHe genome was composed of an open reading frame (ORF, 10296 nucleotides) and 5 'end non coding region (5' LITR, 95 nucleotides) and 3 'terminal non coding region (3' UTR, 585). Nucleotide sequence and amino acid sequence analysis showed that the relationship between the WHe strain and the P3 strain was closest, but the two grade structure of 3 'UTR. was the most similar to the two grade structure of the Vellore P20778 strain 3' UTR.; compared with the other members of the family of the family yellows, the two structure of the most terminal 84 nucleotides of 3 'UTR was rather conservative.
The membrane protein (E) of WHe and P3 plants has three specific amino acids, M (76), G (306) and L (408), while in the other 19 strains, JEV is T (76), E (306), S (408), presumably associated with the host and tissue basophilia of the virus. The phylogenetic map constructed by the whole genome sequence and the phylogenetic map constructed by the E gene have only a few subtle differences, indicating that the E gene is of great significance in the study of the evolutionary relationship among the isolates; 140 JEV E genes from different time and ground regions are transferred from GenBank, and the system has been constructed. Fertility map.
Long chain RT-PCR amplification of full length cDNA of 2.JEV/WHe strain
The long chain RT-PCR technique was used to amplify the genome length eDNA. for PCR amplification, and the amplified fragment of the 3 'terminal and 5' terminal non coding region containing complex two stage structure was sequenced. The amplification results showed that the total length of the genome length of the amplified JEV WHe strain was near 11KB size: the non coding region sequencing analysis showed that the expansion of the genome was increased. It is unique to WHe strain. It is proved that long chain RT-PCR method can be used to amplify the full-length cDNA of genome of JEV W-He strain.
Construction of DNA vaccine vector of 3.JEV/WHe strain
The whole gene of prM/E and NSl was amplified by RT-PCR. and sequence analysis was carried out. It was cloned into eukaryotic expression vector pAVXI., the expression vector pAVXlprM/E and pAVXl-NSl were constructed, and the enzyme digestion and sequence analysis showed that the JEV/WHe strain DNA vaccine vector was constructed successfully.
Expression of NSl gene of 4.WIte strain in Escherichia coli.
The NSl gene was cloned into the pET28b (+) expression vector, and the recombinant expression vector of pET28b (+).NSl was constructed, and the host Escherichia coli BL21 (DE3) was transformed and expressed. The expression was induced and the expression products were detected. The results showed that the relative molecular mass of the expression product was about 43ku. for the development of JE subunit vaccine and DNA vaccine.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373;R392

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