摘要
目的:扩增人幽门螺杆菌(Helicobacter pylori,Hp)热休克蛋白A(heat shock protein A,HspA)编码基因并构建其重组表达载体,进行核苷酸序列分析,并在E.coli BL21中表达,为疫苗的开发奠定了基础。方法:利用PCR技术从Hp基因组DNA中扩增热休克蛋白A(HspA)基因片段,目的基因经SacⅠ和XhoⅠ双酶切、纯化后,插入pET32a(+)载体。以含目的基因片段的重组载体转化大肠杆菌BL21并表达。结果:经PCR、酶切、测序分析表明,插入的基因片段为Hp HspA编码基因,与GenBank报道的相比较,核酸同源性达98%。经SDS-PAGE分析,表达的融合蛋白分子量约为33 KD,其中目的蛋白的分子量约为13 KD。结论:成功地克隆并表达了Hp HspA编码基因,为HspA蛋白质疫苗的研制和快速诊断试剂盒的开发奠定了良好的基础。
Objective： To amplify heat shock protein A gene （HspA） of human helicobacter pylori （Hp）, construct its recombi- nant expression vector, and express HspA in E. coli BL21. Methods： The HspA gene, amplified from Hp by polymerase chain reac- tion （PCR）, was digested by restriction enzyme Sacl and Xho 1 , purified, and inseted into pET32a（ ＋ ） vector. This recombinant vector with target gene segment was confirmed by gene sequence analysis first, then transformed and expressed in Ecoli BL21. Re sults ： Cene sequence analysis showed that the target gene, HspA gene, had been inserted into the recombinant vector. The homology between our target gene sequence and the gene sequnce reported by GenBank is about 98%. SDS - PAGE analysis showed that the HspA fusion gene was successfully expressed in Ecoli BL21. The molecular mass of protein expressed by fusion gene is 33 kD, andthat of target gene is 13 kD. Conclusion： Recombinant expression vector with HspA gene from Hp was successfully constructed, and the HspA fusion protein was successfully expressed, which lays foundation for the development of HspA protein vaccine and a quick diagnosis kit for detection of Hp infection.
出处
《长治医学院学报》
2009年第3期172-175,共4页
Journal of Changzhi Medical College
关键词
幽门螺杆菌
热休克蛋白A
重组载体
基因表达
Helicobacter pylori （Hp）
Heat shock protein A gene （HspA）
Recombinant vector
Gene expression
