根癌土壤农杆菌Ti质粒VirE2基因的克隆及原核表达

Cloning and Expression of the VirE2 Gene of Agribacterium tumefaciens

目的:获得根癌土壤农杆菌Ti质粒VirE2基因原核表达蛋白,为研究其功能打下基础。方法:利用PCR方法,根据发表的根癌土壤农杆菌(Agribacterium tumefaciens)C58Ti质粒基因序列,设计1对引物扩增了C58根癌土壤农杆菌Ti质粒毒性区蛋白VirE2基因,连接T载体经测序,结果与发表的序列同源性达100%。将目的片段从T载体上切下,连接至原核表达载体PET-30a上,转化E.coli DH5α,筛选出阳性克隆,提质粒转化E.coliBL21,用IPTG诱导表达。结果:经SDS-PAGE电泳,发现与对照比较在略小于66kDa处有特异条带,与理论大小(63.5kDa)相符。结论:克隆的VirE2基因获得了原核表达。

Objective：Express the VirE2 gene of Agribacterium tumefaciens and make a foundtion on studying the function of it＇s protein.Method：According to the Ti plasmid gene sequences of Agribacterium tumefaciens published by U.Washington we design a pair of primers,amplified the VirE2 gene situating in Virus region of Agribacterium tumefaciens Ti plasmid with the method of PCR.After connecting the VirE2 gene with T vector,and sequening it we find that there are 100% same with the publishedgene sequence.Cuting the aim fragment from T-vector and joining it to the express vector PET-30a then directed it to the E.coli DH5α,we celected the positive clone,extract their plasmids and directed the plasmids to the E.coli BL21.Inducing it＇s expression by IPTG.Result：through SDS-PAGE elec trophoresis we find there is a distinctive band near 66kD,which identical to theoretical size.Conclusion：The cloned VirE2 gene has been expressed.