摘要
目的克隆幽门螺杆菌(Helicobacter pylori,Hp)26695、J99菌株中粘膜接触诱导因子(induced by contactwith epithelium,iceA)等位基因的iceA1、iceA2基因片段,并将其转入大肠埃希菌中,用IPTG诱导表达目标蛋白,为Hp相关感染菌型诊断奠定基础。方法根据标准菌株Hp 26695、Hp J99的iceA不同基因亚型的开放阅读框设计引物,用PCR方法分别从Hp 26695、Hp J99菌株的基因组DNA中扩增iceA1、iceA2基因片段,分别插入表达载体pET28a和pGEX-4t-1,构建重组表达载体pET28a/iceA1和pGEX-4t-1/iceA2,经序列测定鉴定后,转化大肠埃希菌BL21(DE3)感受态细胞,IPTG诱导重组质表达目标蛋白。表达蛋白经SDS-PAGE、飞行时间质谱鉴定。结果成功构建了重组表达载体pET28a/iceA1和pGEX-4t-1/iceA2,转入大肠埃希菌中,经IPTG诱导后,SDS-PAGE和飞行时间质谱鉴定证实,表达目标蛋白量分别占细菌总蛋白的16.1%和14.2%。结论成功构建了可高效表达IceA1和IceA2蛋白的重组表达载体,为进一步开展相关疾病的诊断研究奠定了基础。
Objective This study sought to clone two genes,iceA1 and iceA2,that are alleles in Helicobacter pylori（H.pylori） strains Hp 26695 and Hp J99,in order to help identify types of bacteria in H.pylori-related infections Methods Primers were designed in accordance with the open reading frames of allelic genes.PCR products were inserted into expression vectors pET28a and pGEX-4t-1,respectively.The recombinant vectors pET28a/iceA1 and pGEX-4t-1/iceA2 were confirmed by sequencing and transformed into E.coli BL21（DE3） competent cells to express target proteins induced by IPTG.The expressed proteins of iceA1 and iceA2 were identified by SDS-PAGE and MALDI-TOF mass spectrometry.Results The recombinant expression vectors pET28a/iceA1 and pGEX-4t-1/iceA2 were successfully constructed.Target proteins were found to account for 16.1% and 14.2% of the total protein in the strains.Conclusion The recombinant expression vectors pET28a/iceA1 and pGEX-4t-1/iceA2 were successfully constructed,and this work should help with further studies of related diseases.
出处
《中国病原生物学杂志》
CSCD
北大核心
2012年第10期743-746,共4页
Journal of Pathogen Biology
基金
"十二五"国家科技支撑计划项目(No.2012BAI06B02)
