基于戊型肝炎病毒中和表位的基因免疫表达载体的构建与鉴定-东南大学学报医学版

基于戊型肝炎病毒中和表位的基因免疫表达载体的构建与鉴定

单位：东南大学基础医学院,病原生物学和免疫学系,江苏,南京,210009

分类号：R512.63, R392.2

出版年·卷·期（页码）：2004·23·第二期（71-74）

摘要：

目的:获得戊型肝炎病毒(HEV)中和表位p166的编码基因,构建基于该中和表位的HEV基因免疫的真核表达载体.方法:应用RT-PCR方法从粪便标本中扩增获得HEV ORF2中G6461～G7035片段,通过比较核酸序列同源性鉴定其基因型,PCR方法扩增该片段中HEV中和表位p166的编码基因,经酶切后克隆于真核表达载体pTR421质粒,并以PCR、酶切和DNA测序加以鉴定.结果:成功克隆了p166的编码基因,其基因型别为Ⅳ型;经酶切鉴定和DNA测序证实p166的编码基因已成功插入pTR421质粒,且核酸序列、读码框架正确.结论:成功构建含Ⅳ型HEV中和表位编码基因的重组质粒,为后续基于HEV中和表位基因免疫的研究奠定基础.

Objective To construct an eukaryotic expression vector used for gene immunization based on HEV neutralization epitope (p166).Methods The gene encoding HEV neutralization epitope was obtained from stool specimens by reverse transcript polymerase chain reaction (RT?PCR) and sequenced and analyzed for HEV genotype.The gene was finally cloned into expression vector-pTR421 after digested by restriction endonucleases.Results The targeted gene was successifully amplified and clustered into HEV genotype IV.The recombinant eukaryotic expression vector (pTR421 p166) based on this gene was constructed and identified as the same as designed originally by using restriction analysis and DNA sequencing.Conclusion An eukaryotic expression vector containing HEV neutralization epitope (p166) encoding gene has been successfully constructed.

参考文献：

[1] Emerson S U, PURCELL R H. Hepatitis E virus. 2003(3). doi:10.1002/rmv.384
[2] KAMILI S, SPELBRING J, KRAWCZYNSKI K. DNA vaccination against hepatitis E virus infection in cynomolgus macaques. 2002(Suppl 3). doi:10.1046/j.1440-1746.17.s3.29.x
[3] 吕冬梅, 陈尔佳, 谢天宏. 戊型肝炎病毒DNA免疫的研究. 中国病毒学2001(2)
[4] MENG J H, DUBREUIL P, PILLOT J. A new PCR-based seroneutralization assay in cell culture for diagnosis of hepatitis E, 1997(6)
[5] Meng J, DAI X, CHANG J C. Identification and characterization of the neutralization epitope(s) of the hepatitis E virus. 2001. doi:10.1006/viro.2001.1093
[6] 萨姆布鲁克J, 拉塞尔D W. 分子克隆实验指南, 2002
[7] 徐薇, 熊思东. 增强以表位为基础的基因免疫效果的新策略. 上海免疫学杂志2002(1). doi:10.3969/j.issn.1001-2478.2002.01.022
[8] 董庆明, 庄辉. 戊型肝炎病毒基因型的研究进展. 中国公共卫生2001(5)
[9] ASHOK M S, RANGARAJAN P N. Protective efficacy of a plasmid DNA encoding Japanese encephalitis virus envelope protein fused to tissue plasminogen activator signal sequences:studies in a murine intracerebral virus challenge mode. 2002(11-12). doi:10.1016/S0264-410X(1)00492-3

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录