中文摘要：

目的克隆幽门螺杆菌毒素相关基因A（cytotoxin-associated gene A,CagA）全长序列并进行序列分析,构建表达CagA的真核表达载体,研究CagA调控胃泌素基因的表达。方法PCR扩增出CagA基因全长片段后构建克隆载体pMD18-T/CagA7和pMD18-T/CagA9,测序结果登录GenBank,登录号分别为GQ161098（NCTC11637）和GQ161099（NCTC11639）,用测序后筛选的PstI和BamHI内切酶将原核载体上的CagA基因酶切后连接到pcDNA3.1/ZEO（-）真核表达载体上,构建真核表达载体pcDNA3.1ZEO（-）/CagA7和pcDNA3.1ZEO（-）/CagA9,并经双酶切和CagA PCR扩增鉴定,最后转染胃癌细胞,检测胃癌细胞中胃泌素基因的表达。结果序列比对结果显示NCTC11637和NCTC11637 CagA的同源性为88.77%,NCTC11637与已收录的NCTC11637（AB015416）同源性为99.22%,序列分析显示两者均有两个Ca-gA酪氨酸磷酸化位点,转染细胞后CagA上调胃泌素基因的表达。结论成功克隆了CagA全长基因,并构建了含CagA全长基因的真核表达载体,在胃癌细胞内上调了胃泌素基因的表达。

英文摘要：

To clone and sequence Helicobacter pylori （H. pylori） cytotoxin-associated gene A （CagA） and construct the eukaryotic expression plasmid in order to study CagA regulatory function for gastrin,H. pylori NCTC11 637 and NCTC11 639 CagA full-length sequences were synthesized by PCR and cloned into pMD18-T plasmid to construct recombinant plasmid pMD18-T/CagA7 and pMD18-T /CagA9. The restriction enzyme PstI and BamHI were used to digest pMD18-T/CagA. The CagA was connected to the pcDNA3.1/ZEO （-） eukaryotic expression vector to construct the eukaryotic expression vector pcDNA3.1ZEO （-） / CagA7 and pcDNA3.1ZEO （-） / CagA9, which then transfected into gastric cancer AGS cells to detect gastrin expression. The GenBank accession number was GQ161 098 for NCTC11 637 and GQ161 099 for NCTC11 639, respectively. It was found that the homology between NCTC11 637 and NCTC11 639 was 88.77% and the homology between NCTC11 637 and published NCTC11637 （AB015416） was 99.22%. Sequence analysis showed that there were two tyrosine phosphorylation sites in CagA, and the gastrin expression was up-regulated after transfecting into AGS cells. The results suggests that CagA full-length gene can be successfully cloned. While PCDNA3.1E（-）/CagA7 and pcDNA3.1ZEO （-）/CagA9 eukaryotic expression vectors are constructed and the gastrin gene expression in transfected AGS cells are up-regulated.