霍乱弧菌O1群和O139群nhaA基因的克隆和变异性研究

目的 克隆霍乱弧菌O1群和O139群nhaA基因，并分析其变异性。方法收集我国1988—2000年散发霍乱弧菌O1群和O139群40株，用聚合酶链反应(PCR)扩增nhaA基因，克隆至pcDNA3载体，通过序列测定分析其同源性和变异性。结果 分别从霍乱弧菌Ol群和O139群扩增出约1．2kb的nhaA基因片段，基因序列分析表明，我国霍乱散发O1群和O139群的nhaA基因与Genbank中霍乱弧菌O1群野毒株参考序列同源性分别为99％和96％。编码氨基酸的突变率分别为2％和11％，nhaA基因重要的结构和功能决定簇(Aspl33、Aspl63、Aspl64、His225、Leu73和Gly338)未发生变异；第203、221位的氨基酸，O1群和O139群发生相同的变异。结论 nhaA基因编码氨基酸的变异可能是霍乱弧菌适应外环境变化的结果。

Clone and mutagenesis analysis of nhaA gene in Vibrio cholerae O1 and O139

Objective Clone and mutagenesis analysis of nhaA gene in Vibrio cholerae O1 and O139. Methods 40 strains of Vibrio cholerae O1 and O139 were collected. A full length nhaA gene fragment was amplified with PCR and cloned into plasmid vector pcDNA3. Homology and mutagenesis of nhaA gene in Vibrio cholerae O1 and O139 were analyzed after sequencing the nhaA gene. Results nhaA gene of Vibrio cholerae O1 and O139 were successfully amplified and cloned. Sequence analysis manifested that nhaA gene of Vibrio cholerae O1 and O139 in China share a high homology with reference sequence of wide-type Vibrio cholerae O1 in GENEBANK(99% and 96% respectively). The amino acid mutagenesis rates of nhaA gene in Vibrio cholerae O1 and O139 were 2% and 11% respectively. The important residues(Asp133,Asp163,Asp164,His225,Leu73 and Gly338 )had no mutation. But in residues 203 and 221 of nhaA gene Vibrio cholerae O1 and O139 had common mutation. Conclusions Mutagenesis of nhaA gene and NhaA protein may be the result of Vibrio cholerae adaptation to survival environment.