摘要
将番茄斑萎病毒(Tomato spotted wilt virus,TSWV)全长外壳蛋白基因亚克隆到原核表达载体pET-32a中,经限制性酶切分析及测序结果表明插入方向正确且阅读框架无移码突变.将重组表达质粒转化大肠杆菌BL21(DE3),SDS-PAGE分析结果证实IPTG可诱导1个分子量约为48 kDa的融合蛋白表达.利用6×His标签单抗和TSWV FoPaTs1多抗证实所表达的蛋白为TSWV外壳蛋白.以纯化的重组外壳蛋白免疫小鼠,制备杂交瘤细胞培养上清单抗,并用杂交瘤细胞培养液上清建立了可靠、有效检测TSWV的Dot-blot ELISA方法.
The full coat protein gene of Tomato spotted wilt virus （TSWV） was subcloned into a prokaryotic expression vector pET-32a. Results of restriction enzyme digestion and DNA sequencing show that the insertion direction and sequence were correct and no frameshift mutation existed. This recombinant prokaryotic expression vector （pET-32a TSWV-CP） was used to transform E. call BL21 （DE3）. SDS-PAGE analysis confirmed that the recombinant coat protein with the predicted molecular weight of about 48 kDa was successfully expressed in E. call BL21 （DE3） strain with the induction of IPTG in the form of a fusion protein. The expressive coat protein was confirmed by 6× His tag monoclonal antibody and TSWV antiserum raised against TSWV FoPaTsl. The purified recombinant coat protein was used to immunize mouse for production of monoclonal antibody. Using culture supernatants of the hybridoma cell lines, a Dot-blot ELISA method was established for reliable and efficient detection of TSWV in plant samples.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2008年第6期597-601,共5页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
上海市科委资助项目(07DZ05020)
