幽门螺杆菌热休克蛋白A亚单位编码基因的克隆与序列分析

目的 幽门螺杆菌(Helicobacter pylori)热休克蛋白A亚单位的编码基因(hspA)克隆和序列分析，为H.pyiori基因工程疫苗的研究奠定基础。方法 应用PCR方法获得国内分离H.pylori菌株MEI，HP27和国际标准参考株H.pylori的hspA基因，通过定向克隆的方法分别插入克隆载体pNEB193中，用质粒酶切电泳和特异PCR方法鉴定重组质粒。克隆基因经测序后进行核苷酸和氨基酸的同源性比较。结果 重组质粒经双酶切后得到0．35kb的hspA基因片段，特异PCR可扩增出hspA基因片段，证实H.pylori hspA基因的重组克隆质粒构建成功。经测序，国内分离HpMEL-HP27的hspA基因全长357bp(Genbank收录号：AY295084)，编码由118个氨基酸残基组成的肽链，hspA基因序列与GenBank公布的H.pylorl相应基因同源性高达95．20％～97．48％，氨基酸序列同源性在95．76％～97．46％之间。结论 克隆了H．pylori菌株MEL-HP27的hspA基因，其核酸序列与国际参考株NCTC11637同源性为97．48％。

Cloning and sequence analysis of heat shock protein subunit A gene of H.pylori

Objective To clone and sequence hspA gene of H. pylori, and to supply basis on genetic engineering vaccine of H. pylori. Methods hspA gene was amplified by PCR from clinical strain MEL-HP27 and international reference strain NCTC 11637 chromosome DNA. hspA gene fragments were inserted into the clone vector pNEB193, and then were transformed into E. coli JM109. The recombinant plasmids were identified by restriction fragments electrophoresis and specific PCR. The sequence of hspA gene was determined and analyzed by biological software Omiga2 .0. Results The recombinant plasmid was digested into two fragments which are clone vector fragment and hspA gene fragment. 0.35kb hspA gene fragment could be amplified by specific PCR. It demonstrated that recombinant plasmids contained hspA gene. Gene sequencing results showed that hspA gene fragment was 357bp long. hspA sequence of MEL - HP27 had high homology from 95.20% to 97.48% as compared to those reported in Genbank and amino acids homology was from 95. 76% to 97. 46%. Conclusion HspA gene of H.pylori MEL-HP27 was cloned successfully,and its nuclear sequence had homology of 97.48% compared with international standard reference H. pylori NCTC 11637.