日本脑炎病毒(JEV)内蒙古分离株M73-1E基因的5′端克隆及部分序列分析

Cloning and Nucleotide Sequence of E gene 5′ End of Japanese Encephalitis Virus NeiMongol Isolate JEV M73-1

根据国外报道的 JEV( Japanese encephalitis virus)基因组全序列 ,针对 JEV E基因 5′端设计合成一对特异引物 ,以日本脑炎病毒内蒙古分离株 M73-1 RNA为模板 ,经 RT-PCR扩增 ,获得 366bp的 JEV E基因 c DNA片段 .将此片段重组于质粒 p UC1 9中 ,并转化大肠杆菌DH5α.经 PCR扩增 ,酶切及序列分析 ,结果表明 JEV M73-1 5′端 1 2 6个核苷酸序列与 JEV日本 Nakayama株和 Ja OAr S982株的同源性分别为 96.8%和 96.8% ,氨基酸序列同源性均为 99.2 % .通过对病毒基因组结构分析从分子水平上进一步证实了 1 973年在呼和浩特市流行的脑炎是由 JEV引起的流乙脑炎 .E基因编码 JEV的囊膜糖蛋白 ,是其重要表面抗原 .JEV M73-1 E基因 5′端克隆和部分序列分析对 JEV的分子生物学研究。

A 5′ end cDNA fragment 366 bp in length of E gene of Japanese encephalitis virus(JEV) Inner Mongolia isolate M73 1 was synthesized and amplified by RT PCR with a pair of primers designed according to the references. This cDNA fragment was inserted into plasmid pUC 19 at XbaI and BamHI sites and cloned in E.coli DH5α. PCR amplification,restriction endonuclease digestion and partial sequence analysis show that a recombinant pUC 19 named pJV366 was obtained. The sequence analysis of 5′ end 126 nucleotides of E gene of JEV M73 1 revealed that the nucleotide sequence homology between JEV M73 1 and Japanese strains JEV Nakayama and JaOArs 982 was 96.8% and 96.8% respectively and the deduced amino acid sequence was both 99.2%. So it was further confirmed at molecular level that the epidemic encephalitis in 1973 in Hohhot was Japanese encephalitis caused by JEV. Molecular cloning and partial sequence analysis of 5′ end of E gene are of theoretical and practical importance for further molecular biology study on JEV M73 1, its nucleic acid based diagnosis and preparation of genetic engineering vaccine.