麻疹病毒HA基因克隆和表达的研究  

Study on cloning and expression of measles virus hemagglutinin gene

作　　者：姚栩[1] 陈智伟[1] 刘树韬[2] 黄晓霞[1] 

机构地区：[1]福州市疾病预防控制中心,福建福州350005 [2]福州大学生物工程与科学学院,福建福州350009

出　　处：《中国卫生检验杂志》2013年第11期2481-2483,2486,共4页Chinese Journal of Health Laboratory Technology

基　　金：福州市科技局(2009-S-93)

摘　　要：目的构建麻疹病毒血凝素蛋白基因的重组质粒,使其在大肠埃希菌中表达HA蛋白,为麻疹病毒诊断奠定基础。方法从麻疹病毒株MV07-30中提取核糖核酸,用RT-PCR扩增HA基因,用限制性内切酶BamHⅠ和HindⅢ双酶切HA基因片段和pGEM-Teasy载体,连接后获得重组质粒pGEM-Teasy-MVHA并克隆到大肠杆菌表达载体pET28a(+);用IPTG诱导重组质粒在大肠杆菌BL21(DE3)中进行表达;利用SDS-聚丙烯酰胺凝胶电泳(PAGE)技术进行重组蛋白表达的分析;通过Ni-NTA亲和层析获得高纯度目的蛋白。结果 RT-PCR获得的HA基因片段约长1700 bp。转化的大肠杆菌表达产物在SDS-PAGE中出现与目的蛋白分子量相一致的条带,且重组蛋白主要以包涵体形式存在;血凝及血凝抑制实验显示表达产物具有良好的抗原性。结论构建麻疹病毒HA基因的原核表达体系,纯化的重组蛋白可用作麻疹病毒检测的抗原。Objective To construct recombinant plasmid pGEM - Teasy - MVHA and express the hemagglutinin gene of mea- sles virus in Escherichia coli expression system, in order to lay a foundation for diagnosis of measles virus. Methods Measles virus genomic RNA was extracted from the strain MV07 - 30, the interest HA gene was amplified by RT - PCR. After digested by Bam - H I and Hind - m enzymes,the pGEM - Teasy - MVHA was inserted into expression plasmid pET28a（ ＋ ） vector, the recombinant plasmid was transformed into E. coli BL21 （ DE3 ） cells and induced by IPTG. The expression of HA protein was an- alyzed by SDS - PAGE. Then the recombinant protein was purified with the Ni - NTA column. Results The interest gene am- plified by RT- PCR was 1700 bp. The interest protein was expressed in inclusion in Escherichia coli expression system, and showed the high antigenicity in Western blotting. Conclusion The interest protein was successfully expressed in prokaryotic system, and can be used as the antigen in measles virus detection after purification.

关 键 词：麻疹病毒 血凝素 基因克隆 原核表达 

分 类 号：R511.1[医药卫生—内科学]