幽门螺杆菌尿素酶B基因的限制性片段长度多态性分型和生物信息学分析

PCR-RFLP and bioinformatic assay of urease B gene from different helicobacter pylori strains

目的：对不同来源野生型幽门螺杆菌（Hp）菌株的尿素酶B基因进行限制性片段长度多态性（RFLP）分型和生物信息学分析。方法：采用HaeⅢ单酶切法对国内临床分离的Hp菌株、动物模型适应株及国际标准Hp菌株尿素酶B（ureB）基因进行RFLP分型，同时进行基因测序和序列同源性对比及进化树分析等生物信息学研究。结果：HaeⅢ单酶切结果显示，14株Hp 1．7kb ureB基因均呈现出2～5种带型，可分为五组，其中两种国际标准株NCTC11637、NCTC11639和三种临床分离野生株拥有相同的RFLP带型，其他国内临床分离株和动物适应株分属于四种不同的RFLP带型组。ureB基因序列同源性比对表明，各种Hp菌株间核苷酸序列同源性均〉96．6％，氨基酸序列同源性均〉98％，其中CCS9801、CCS9806、M3及M10菌株之间的核苷酸和氨基酸序列同源性为100％。核苷酸序列进化树分析显示，2株国际标准株与国内临床分离株及动物适应株分处于不同进化分支，为平行进化关系；而在氨基酸序列进化树中转变为非平行关系。结论：不同Hp菌株尿素酶B在基因和蛋白质水平均高度保守和同源。

Objective ：To analyze the polymorphism of urease B gene （ureB） from different Hp strains. Methods：The sequences of ureB gene from 14 different Hp strains were amplified by PCR to analyze PCR-RFLP with HaeⅢ digestion, At the same time, the sequence of ureB gene was analyzed with biochemical software to compare the homology of nucleotide and amino acid sequence and to profile the phylogenetic tree. Results ： The results of 1.7 kb ureB gene digested with HaeⅢ showed there were 2 - 5 band types in 14 different Hp strains and formed five distinct RFLP types. The two reference strains had the same RFLP type as 3 clinical isolates while the other clinical isolates and 3 isolates from animal model belong to 4 different RFLP types respectively. The nucleotide sequences and putative amino acide sequences of Hp ureB were compared. The various ureB sequences had high homologies （ more than 96. 6% and 98% in nucleotide sequences and amino acide sequences respectively） among 14 Hp strains. Particularly, there was the highest homologies （ 100% ） between CCS9801 ,CCS9806 ,M3 and M10. Phylogenetic tree analysis showed that two reference strains and other isolates from clinical patients and Hpinfected mice model were located in two different lineage respectively in phylogenetic tree of nucleotide sequences while there were some variance in phylogenetic tree of amino acide sequences. Conclusion ： Hp ureB was high conserved and homologous in the gene level as well as in the protein level .