摘要
目的 :获取含人幽门螺杆菌 (Hp)热休克蛋白A编码基因并构建其重组载体 ,进行核苷酸序列分析 ,为在E .coliBL2 1中表达 ,疫苗的开发奠定基础。方法 :利用分子克隆技术从HpDNA染色体中 ,扩增热休克蛋白A编码基因片段。将目的基因与pET32a(+)同时经kpnⅠ、BamHⅠ双酶切、纯化、连接后。转化含有目的基因的重组载体 ,进行序列分析。 结果 :经酶切、测序分析表明 ,插入的基因片段为Hp热休克蛋白A编码基因 ,与GenBank的报道相比较 ,有 1.4 %的bp发生变异 ,1.6 %的氨基酸残基改变。其同源性高达 98%。结论 :成功地克隆了Hp热休克蛋白A编码基因 ,为Hp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 obtain DNA of human Helicobacter pylori heat shock protein A, and construct a recombinant vector containing gene encoding HspA for nucleotide sequence analysis.Methods:The target gene was amplified from Hp chromosome by PCR, and then digested by restricted endonuclease enzyme of kpn I , BamH I simultanously, and inserted into the prokaryotic expression vector pET32a(+) digested by corresponding restricted endonuclease enzyme. The recombinant vector was selected and transformed for nucleotide sequence analysis.Results:Enzyme digestion analysis and sequencing showed that the target gene had been inserted into recombinant vector, but as compared with gene reported by GenBank. 1.4 % of the gene mutation and 1.6% of amino acid residues change in Hp happened respectively. The DNA sequence analysis showed the sequence of HspA DNA was almost the same as that published by GenBank.Conclusion:The gene coding for Hp HspA is cloned successfully.The results obtained lay the foundation for research on development of Hp protein vaccine and a quickly diagnostic kit applying to detection of Hp infection.
出处
《重庆医科大学学报》
CAS
CSCD
2003年第4期414-416,420,共4页
Journal of Chongqing Medical University
关键词
幽门螺杆菌
热休克蛋白A
编码基因
重组载体
疫苗
Helicobacter pylori
Heat shock protein A
Encoding gene
Recombinant vector
Vaccine
