幽门螺杆菌vacA基因毒性相关片段的克隆及序列分析  

Cloning and sequence analysis of a fragment related to vacuolating cytotoxin of vacA of Helicobacter pylori

作　　者：江红[1] 阎小君[1] 苏成芝[1] 韩锋产[1] 冯永强 侯瑜 

机构地区：[1]中国人民解放军第四军医大学基因诊断技术研究所,陕西省西安市710032

出　　处：《世界华人消化杂志》2000年第7期728-732,共5页World Chinese Journal of Digestology

摘　　要：目的 vac A 基因编码的蛋白是幽门螺杆菌的一个重要毒力因子,通过空泡化作用损伤上皮细胞.vacA 基因与 Hp 感染者的临床发病有着密切的关系,vacA 的基因型决定毒素蛋白体外表达水平的高低.我们从幽门螺杆菌88022菌株中扩增出vacA 基因毒性相关片段,进行序列测定和序列比较分析,为Hp 的临床检测奠定基础.方法以该菌株总 DNA 为模板,紧随 vacA 基因序列 s 区保守区域的引物(位于316bp～335bp 和1198bp～1218bp)扩增vacA 基因的一个903bp 片段,编码的氨基酸为34～334位,用13amHI 和 Pst Ⅰ双酶切后克隆入 pGEM-3Zf(-)质粒载体,以双脱氧法双向测定目的片段的序列,拼接出该片段的全序列,并与已知的 Hp vacA 基因序列作比较.结果所得序列与 Hp 国际标准株 CCUG17874(GeneBank gi619248)和 NCTCll638(GeneBank gi 495469)的 vacA 基因序列完全一致,氨基酸序列分析和所报道的结果一致.结论构建了 Hp vacA 基因毒性相关片段的重组克隆,为以后的进一步研究奠定了基础.AIM We clone and sequence a fragment related to vacuolating cytotoxin of vacA from Helicobacter pylori (Hp) strain 88022 for the clinical analysis of Hp vacA. METHODS A 903 bp fragment was amplified by the polymerase chain reaction.The template DNA was purified from Hp strain 88022.We designed the primers located in 316bp-335bp and 1198bp-1218bp respectively,from the relatively reserved region of vacA gene.DNA fragment containing the sequence codes for amino acids 34-334,was digested by BamH Ⅰ and Pst Ⅰ and cloned into pGEM-3Zf(-) plasmid.Its sequence was determined with an autosequencing instrument from two directions and compared with those having the corresponding region of vacA from other Hp strains. RESULTS The DNA sequence we got in this study is the same as that of Hp strain CCUG17874 (GeneBank gi 619248) and NCTCl1638 (GeneBank gi 495469). CONCLUSION We got a recombined clone of Hp vacA related to vacuolating cytotoxin which has laid the foundation for further studies.

关 键 词：幽门螺杆菌 VACA基因 聚合酶链反应 克隆 

分 类 号：R450[医药卫生—治疗学]