本文選題：日本腦炎病毒 + WHe株��； 參考：《西北農(nóng)林科技大學(xué)》2007年碩士論文

【摘要】： 日本腦炎(Japanese Encephalitis,JE),又稱流行性乙型腦炎,是由日本腦炎病毒(Japanese Encephalitis Virus,JEV)引起的一種中樞神經(jīng)系統(tǒng)人畜共患急性傳染病。其致死率高達(dá)30%左右,50%的患者會(huì)留下永久性的后遺癥,其中,中國的JE發(fā)病數(shù)占世界總發(fā)病數(shù)的80%以上。庫蚊伊蚊是該病的主要傳播媒介,而豬則是重要的傳染源和放大器。豬患JE的主要表現(xiàn)為懷孕母豬流產(chǎn)、產(chǎn)死胎、弱仔、公豬睪丸炎及少數(shù)仔豬呈神經(jīng)癥狀,給養(yǎng)豬業(yè)造成了巨大的經(jīng)濟(jì)損失。目前,用于JE免疫預(yù)防的疫苗有滅活疫苗和弱毒疫苗兩種,其安全性和較高的生產(chǎn)應(yīng)用成本已成為人們?nèi)找骊P(guān)注的問題。近年來,病毒分子生物學(xué)和反向遺傳學(xué)的發(fā)展為開發(fā)新型JE疫苗提供了新的技術(shù)手段。 本研究為進(jìn)一步探索JEV/WHe株基因組功能、致病機(jī)理及構(gòu)建高效新型疫苗,開展了以下工作: 1.JEV/WHe株全基因組特征分析 本研究設(shè)計(jì)了11對(duì)特異性引物,采用RT-PCR技術(shù)擴(kuò)增得到覆蓋JEV/WHe株基因組全長(zhǎng)cDNA的11個(gè)特異性片段,測(cè)序分析表明,JEV/WHe株基因組由一個(gè)開放閱讀框(ORF,10296個(gè)核苷酸)和5’端非編碼區(qū)(5’LITR,95個(gè)核苷酸)和3’端非編碼區(qū)(3’UTR,585個(gè)核苷酸)組成。核苷酸序列和氨基酸序列分析表明,WHe株與P3株親緣關(guān)系最近,但3’UTR.形成的二級(jí)結(jié)構(gòu)與Vellore P20778株3’UTR.形成的二級(jí)結(jié)構(gòu)最相似;與黃病毒科其他屬成員比較,3’UTR最末端約84個(gè)核苷酸所形成的二級(jí)結(jié)構(gòu)相當(dāng)保守。 WHe株與P3株的膜蛋白(E)擁有M(76)、G(306)和L(408)三個(gè)特有氨基酸,而在其它19株JEV為T(76)、E(306)、S(408),推測(cè)可能與病毒的宿主及組織嗜性有關(guān)。WHe株E蛋白中存在控制黃病毒毒力的RGD(387-389)基序,而一部分弱毒株也有該基序,提示JVE毒力型的決定簇具有毒株特異性。由全基因組序列構(gòu)建的系統(tǒng)發(fā)育圖與由E基因構(gòu)建的系統(tǒng)發(fā)育圖僅有一些細(xì)微的差別,表明E基因在研究各分離株之間進(jìn)化關(guān)系中具有重要意義;從GenBank中調(diào)出不同時(shí)間和地區(qū)分離的140株JEV E基因,構(gòu)建了系統(tǒng)發(fā)育圖譜。 2.JEV/WHe株全長(zhǎng)cDNA的長(zhǎng)鏈RT-PCR法擴(kuò)增 采用長(zhǎng)鏈RT-PCR技術(shù)擴(kuò)增基因組全長(zhǎng)eDNA。對(duì)其進(jìn)行PCR擴(kuò)增鑒定,并對(duì)含復(fù)雜二級(jí)結(jié)構(gòu)的3’末端和5’末端非編碼區(qū)擴(kuò)增片段進(jìn)行了測(cè)序分析。擴(kuò)增結(jié)果表明,擴(kuò)增出的JEV WHe株基因組全長(zhǎng)cDNA分子近11kb大小:非編碼區(qū)測(cè)序分析表明,擴(kuò)增產(chǎn)物為WHe株所特有。證明長(zhǎng)鏈RT-PCR法可用于JEV W-He株基因組全長(zhǎng)cDNA的擴(kuò)增。 3.JEV/WHe株DNA疫苗載體構(gòu)建 采用RT-PCR.技術(shù)擴(kuò)增prM/E和NSl全基因并進(jìn)行序列分析,將其分別克隆于真核表達(dá)載體pAVXI.,構(gòu)建的表達(dá)載體pAVXlprM/E和pAVXl-NSl,酶切鑒定和序列分析表明,JEV/WHe株DNA疫苗載體構(gòu)建成功。 4.WIte株NSl基因在大腸桿菌中的表達(dá). 將NSl基因克隆到pET28b(+)表達(dá)載體,構(gòu)建了pET28b(+).NSl重組表達(dá)載體,轉(zhuǎn)化表達(dá)宿主大腸桿菌BL21(DE3),并對(duì)其進(jìn)行了誘導(dǎo)表達(dá),對(duì)表達(dá)產(chǎn)物進(jìn)行檢測(cè)。結(jié)果表明,表達(dá)產(chǎn)物相對(duì)分子質(zhì)量約為43ku。為JE亞單位疫苗和DNA疫苗的開發(fā)奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373;R392

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