乙脑病毒E蛋白的原核表达、纯化及初步鉴定  

Expression, purification and identification of gene encoding JEV E protein in prokaryotic cells

作　　者：高淑娴[1] 张伟[1] 任君萍[1] 雷迎峰[1] 丁天兵[1] 宋建华[1] 马文煜[1] 徐志凯[1] 

机构地区：[1]第四军医大学基础部微生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2007年第16期1459-1461,共3页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30400378)

摘　　要：目的:构建乙脑病毒(JEV)E蛋白重组载体并在原核细胞BL21(DE3)中表达.方法:采用RT-PCR扩增片段,定向克隆入pET28a(+)中;重组载体pET28a-6His-E转化BL21(DE3),通过酶切、SDS-PAGE和Western Blotting检测其载体构建和蛋白表达;表达产物包涵体经Ni-NTA亲和层析纯化.结果:构建得到原核融合重组载体pET28a-6His-E;诱导后表达得到6His-E融合蛋白,纯化得到表达产物并进行了初步的鉴定.结论:表达并纯化得到JEVE蛋白原核表达产物.AIM： To construct gene encoding Japanese encepha- litis virus （JEV） envelope glycoprotein and to express E protein in BL21 （ DE3 ）. METHODS： Using RT-PCR, the gene encoding E protein was amplified. The gene was cloned into pET28a（ ＋ ） and the recombinant plasmid pET28-6His-E was transformed into BL21 （ DE3 ）. The 6His-E protein expressed in BL21 （ED3） was determined by SDS-PAGE and Western Blotting. The expression product in inclusion body was purified by NiNTA chromatography. RESULTS ： Sequencing of E gene revealed that the mutation rate was 0.2% （3/1500） and the base mutation didn＇t change the amino acid nonsense. The recombinant expres- sion vector pET28-6His-E was constructed and the 6His-E protein was successfully expressed in an insoluble form. After expression and purification by metal chelate affinity chromatography, purified 6His-E fusion protein was obtained. CONCLUSION： The E protein can be expressed in prokaryotic cell and would be useful for further research on the receptor of JEV and its infection mechanisms.

关 键 词：脑炎病毒 日本 病毒包膜蛋白质类 重组融和蛋白质类 

分 类 号：R512.32[医药卫生—内科学]