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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 and express the VP3 gene of rotavirus (TB) TB-Chen strain of group A and to make molecular phylogenetic analysis and genotyping. Methods The gene encoding VP3 of human rotavirus TB-Chen strain was amplified by PCR and cloned into pETL vector. The recombinant prokaryotic expression vector pET-VP3 was constructed and transformed into E. coli BL21 (DE3) for expression. The expressed recombinant protein was identified by SDS-PAGE and Western blot, and the VP3 gene was molecularly phylogenetically analyzed. Results The recombinant plasmid pET-VP3 was correctly constructed by double enzyme digestion and sequencing. The recombinant protein was expressed in the form of inclusion bodies with a relative molecular mass of 98 000. The recombinant VP3 protein could be recognized by the anti-SA11 whole virus guinea pig serum. The group A RV VP3 found so far can be divided into 7 genotypes, and the TB3 strain and SA11 strain VP3 gene belong to M2 type and M5 type, respectively. Conclusion The VP3 protein of human RV TB-Chen strain was successfully expressed in prokaryotic cells in group A, which laid the foundation for further study on the structural function of VP3 and the development of new RV vaccines, diagnostic reagents and therapeutic drugs.