幽门螺杆菌中性白细胞激活蛋白基因napA克隆及序列分析

Cloning and sequence analysis of neutrophil-activating protein gene napA of H.pylori

目的 :从幽门螺杆菌 (H .pylori)中克隆中性白细胞激活蛋白基因napA并构建重组载体。方法 :提取H .pylori郑州分离株MEL HP2 7基因组DNA作模板 ;根据H .pyloriJ99全基因组序列设计napAPCR引物 ,高保真酶扩增napA片断 ;限制性内切酶切PCR产物和pNEB193空质粒 ,T4DNA酶连接酶切产物 ,重组质粒转化E .coliTB1工程菌 ,蓝白斑试验筛选阳性克隆 ;提取质粒经酶切鉴定后测序并利用生物学软件进行序列分析。结果 :DNA限制性内切酶可从重组质粒pNEB napA的EcoRⅠ和SalⅠ位点之间切出一个 4 35bp的DNA片断 ,测序结果与J99napA同源性为 96 .5 % ,与H .pylori其他菌株的同源性为 92 %～ 97%。结论 :成功构建H .pylori中性白细胞激活蛋白基因的重组质粒pNEB napA ;序列分析表明HP napA是一类高度保守的原核型基因 ,可作为H .pylori疫苗候选基因。

Aim: To clone neutrophil-activating protein gene of H.pylori and construct the recombinant plasmid carrying HP-napA gene. Methods: Genomic DNA was isolated from clinical strain MEL-HP27 as PCR template , primers were designed according to the genomic sequence of H.pylori J99 published on Genbank, then napA gene was amplified by PCR using pyrobest DNA polymerase. DNA fragment of napA and clone vector pNEB193 were digested by the same restriction endonucleases and linked by T4 DNA ligase. The recombin plasmid was transformed into E.coli TB1.The positive clones were selected by blue-white screening, then identified by the methods of restriction endonuclease digesting and DNA sequencing. Results: A 435 bp DNA fragment was inserted into pNEB193 vector between EcoRⅠ and SalⅠ restriction sits.Compared with H.pylori J99 napA, the homology was 96.5%. Blast on NCBI compared with other H.pylori strainsthe nucleotide acids homology was between 92% and 97%. Conclusions: pNEB-napA ,the recombined plasmid carring neutrophil-activating protein gene has been successfully constructed, and sequence analysis indicates that HP-napA is a highly conserved prokaryotic gene and might be a potential candidate for H.pylori vaccine development.