幽门螺杆菌Ⅳ型分泌系统cagX基因克隆及生物信息学特征预测.docVIP

幽门螺杆菌Ⅳ型分泌系统cagX基因克隆及生物信息学特征预测 　【摘要】 目的： 构建表达幽门螺杆菌（Helicobacter pylori，H. pylori） NCTC 11637 IV型分泌系统cagX(HP0528)全长编码基因的原核表达载体。方法： 用PCR方法从H. pylori基因组DNA中扩增cagX编码基因片段，将cagX克隆至pGEM-T载体，转化大肠杆菌DH5α，再将其定向插入pET32a(+)载体中，经IPTG诱导表达。SDS分析表达结果，并进行序列测定和生物信息学预测。结果： 克隆幽门螺杆菌NCTC 11637 cagX 基因全长1 569 bp（基因库登录号为EF608160），编码522个氨基酸，融合蛋白的相对分子质量约为80.5 kD。与基因库公布的其他H. pylori菌株基因序列的核苷酸同源性为96%～99%。预测结果显示H. pylori 11637，26695，J99间二级结构有一定差异，但抗原性相同。结论： 成功克隆并表达了H. pylori NCTC 11637 cagX 基因，并对其生物学特征进行了预测，为进一步研究其生物学功能奠定了基础。 【关键词】 幽门螺杆菌； cagX； IV 型分泌系统； 生物信息； 序列分析 ［Abstract］ Objective: The prokaryotic expression vector for gene encoding the fusion protein cagX of type IV secretion system(TFSS) was constructed and expressed. Methods： H. pylori cagX gene was amplified by PCR using the genome DNA. The PCR product was inserted into pGEM-T vector and then transformed into E.coli DH5α. The positive recombinant clone was analyzed by digestion of restriction endonuclease. Then the cagX gene fragment was inserted directionally into vector pET32a(+) to construct recombinant clones of cagX.After they were expressed by BL-21 E.coli cells, the PCR products were sequenced and analyzed. Results： A 1569 base pairs long cagX gene, which encodes a product of 522 amino acid, was obtained using PCR method and was cloned into pGEM-T vector successfully. It was found that the relative molecular weight of this fusion protein was approximately about 80.5 kD. GenBank accession number is EF608160.The sequence analysis for cagX showed that it shares 96%～99% homology with other strains in GenBank. The result of analysis revesled that there were certain differences in secondary structure, but the antigenicity was identical. Conclusion： It was indicated that we had obtained the correct cagX gene， which posed a basis for further researching on its biological function. ［Key words］ Helicobacter pylori; cagX; type IV secretion system；bioinformatics；sequence analysis 　　幽门螺杆菌(Helicobacter pylori, H. pylori)是一种定植于人类胃粘膜的螺旋状、革兰阴性微需氧菌