幽门螺杆菌尿素酶B亚单位编码基因的克隆与序列分析

目的 幽门螺杆菌(Helicobacter pylori)尿素酶B亚单位的编码基因(ureB)的克隆和序列分析，为H．pylori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ureB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1．71kb的ureB基因片段，特异PCR可扩增出ureB基因片段，证实H．pylori ureB基因的重组克隆质粒构建成功。经测序，国内分离H．pylori MEL-HP27的ureB基因全长1710bp( Genbank收录号：AY295085)，编码由569个氨基酸残基组成的肽链，ureB基因序列与GenBank公布的H．pylori相应基因同源性高达96．08％～98．30％，氨基酸序列同源性在98．77％．99．82％之间。结论 成功克隆了MEL-HP27菌株的ureB基因，其核苷酸序列与国际参考株NCTC11637的同源性为97．67％。

Cloning and sequence analysis of urease subunit B gene of H.pylori

Objective To clone and sequence ureB gene of H. pylori, and to supply basis on genetic engineering vaccine of H. pylori. Methods ureB gene was amplified by PCR from clinical strain MEL-HP27 and international reference strain NCTC11637 chromosome DNA. ureB gene fragments were inserted into the vector pNEB193,and then were transformed into the E. coli JM109.The recombinant plasmids were identified by restriction fragment electrophoresis and specific PCR.The sequence of ureB gene was determined and analyzed by biological software Omiga2 .0. Results The recombinant plasmid was digested into two fragments which are vector fragment and ureB gene fragment. 1 .71kb ureB gene fragment could be amplified by specific PCR. It demonstrated that recombinant plasmids were contain ureB gene. Gene sequencing results showed that ureB gene fragment was 1710bp long. ureB gene sequence of MEL-HP27 had high homology from 96.08% to 98.30% as compared to those reported in Genbank and amino acids homology was from 98 .77 % to 99.82 % . Conclusion HspA gene of H. pylori MEL-HP27 was cloned successfully,and its nucleotide sequence had homology of 97.48% compared with international standard reference H. pylori NCTC11637.