摘要
【目的】克隆鸡传染性法氏囊病病毒(IBDV)BJ-10株VP2基因,构建其原核表达载体,并在大肠埃希菌中进行高效表达。【方法】根据IBDVVP2基因序列设计1对特异性引物,应用RT-PCR技术克隆IBDV BJ-10株VP2基因;将目的基因插入pMD18-T连接载体,筛选出阳性重组质粒;将该质粒克隆至原核表达载体pET-32α中,转化入JM109,经IPTG诱导表达,对产物进行SDS-PAGE电泳分析,确定IPTG的最佳诱导表达时间和浓度。【结果】克隆到了全长1 356bp的IBDV BJ-10株VP2基因;PCR、酶切鉴定分析表明,成功构建了重组质粒pET-32α-VP2;SDS-PAGE电泳分析表明,在宿主菌JM109中成功表达了约为60ku的VP2蛋白;IPTG诱导表达时间以4.5h为宜,诱导最佳浓度为0.8mmol/L。【结论】成功克隆了IBDV BJ-10株VP2基因,并在大肠埃希菌中表达了VP2蛋白。
[Objective] This study aimed to clone the VP2 gene of infectious bursal disease virus （IB- DV） BJ 10 isolate, construct the prokaryotic expression vectors, and express them in Escherichia coli. [Method] A pair of specific primers were designed according to the sequences of IBDV BJ-10 strain,and the VP2 gene was cloned by RT-PCR. The IBDV VP2 gene was inserted into ligation vector pMD18-T and the recombinant plasmid was selected. The recombinant plasmid was cloned into the prokaryotic expression vector pET32~ and transformed into E. coli JM109. Then it was highly expressed by inducing with IPTG and proved by SDS-PAGE electrophoretic analysis, to designate the best inducement time and concentra- tion. [Result] The IBDV BJ 10 VP2 gene with 1 356 bp was cloned. From the PCR and identification by enzyme digestion analysis, the recombinant plasmid pET 32^-VP2 was constructed. SDS-PAGE analysis in- dicated that the VP2 protein 60 ku in size was expressed properly from E. coli JM109. The best IPTG in- ducement time was 4.5 h,and the best concentration was 0.8 mmol/L. [Conclusion] The IBDV BJ-10 iso- late VP2 gene was successfully cloned,and the VP2 protein was expressed in E. coli.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2013年第9期38-42,共5页
Journal of Northwest A&F University(Natural Science Edition)
基金
渭南市基础研究计划项目(2011JH-6)
