A组人轮状病毒VP3基因的原核表达及鉴定  

Prokaryotic expression and identification of VP3 gene of group A human rotavirus

作　　者：张顺[1] 潘小霞[1] 袁静[1] 文喻玲[1] 陈元鼎[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《中国生物制品学杂志》2012年第2期152-156,共5页Chinese Journal of Biologicals

摘　　要：目的克隆并原核表达A组人轮状病毒(Rotavirus,RV)TB-Chen株结构蛋白VP3基因,并进行分子系统进化和基因分型。方法采用PCR法扩增A组人轮状病毒TB-Chen株VP3编码基因,克隆至pETL载体上,构建重组原核表达质粒pET-VP3,转化大肠杆菌BL21(DE3),并进行表达。表达的重组蛋白经SDS-PAGE及Western blot鉴定后,对VP3基因进行分子系统进化及基因型分析。结果重组表达质粒pET-VP3经双酶切及测序证实构建正确;表达的重组蛋白相对分子质量约为98 000,以包涵体形式存在;重组VP3蛋白可被抗SA11株全病毒豚鼠免疫血清识别;迄今发现的A组RV VP3可分为7个基因型,TB-Chen株和SA11株VP3基因分别属于M2型和M5型。结论成功原核表达了A组人RV TB-Chen株VP3蛋白,为进一步研究VP3的结构功能及开发新型RV疫苗、诊断试剂和治疗药物奠定了基础。Objective To clone the gene encoding structural protein VP3 of group A human rotavirus （RV） TB-Chen strain, express in prokaryotic cells and investigate its molecular phylogenesis and genotype. Methods The gene encoding VP3 of TB-Chen strain was amplified by PCR and cloned into pETL vactor. The constructed recombinant plasmid pET-VP3 was transformed to E. coli BL21 （DE3）. The expressed recombinant protein was identified by SDS-PAGE and Western blot, based on which the cloned VP3 gene was analyzed for molecular phylogenesis and genotype. Results Restriction analysis and sequencing proved that recombinant plasmid pET-VP3 was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 98 000, existed in a form of inclusion body and was recognized by the guinea pig sera against whole virus of SAll strain. By the so far, the discovered RV VP3 was of seven genotypes, of which those from TB-Chen and SA11 strains were belonged to genotypes M2 and M5 respectively. Conclusion The VP3 protein of group A human RV TB-Chen strain was successfully expressed in prokaryotic cells, which laid a foundation of further study on structure and function as well as development of VP3.

关 键 词：轮状病毒属 VP3基因 原核细胞 基因表达 基因分型 

分 类 号：R373.2[医药卫生—病原生物学] Q786[医药卫生—基础医学]