幽门螺杆菌儿童分离株中性粒细胞激活蛋白克隆及表达序列

目的：从幽门螺杆菌临床儿童分离株GZCH1基因组扩增中性粒细胞激活蛋白（NAP）基因,克隆入T载体,并亚克隆入表达载体pGEX-4T-1,进行测序及基因比对分析,为幽门螺杆菌疫苗研制奠定基础.方法：根据GenBank中幽门螺杆菌NAP序列,设计一对特异性引物扩增幽门螺杆菌临床儿童分离株NAP全长基因,与T载体连接,转化大肠杆菌DH5α,提取质粒进行酶切、测序鉴定,经EcoR Ⅰ、Not Ⅰ双酶切后与做相应酶切的pGEX-4T-1连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定,IPTG诱导重组蛋白表达,并对基因进行测序及比对分析.结果：以幽门螺杆菌儿童分离株GZCH1为模板,成功扩增了NAP基因,基因大小为435 bp,重组pGEX-4T-1-NAP双酶切鉴定可见目的片段,测序结果显示NAP在正确读框中,序列比对分析显示其与幽门螺杆菌J99株氨基酸一致性达99.2％,IPTG诱导后,pGEX4T-1-NAP/BL21在相应分子量（42.8 kD）可见融合蛋白的表达.幽门螺杆菌儿童分离株GZCH1 NAP序列已登录GenBank（登录号：GU301881）.结论：从幽门螺杆菌临床儿童分离株GZCH1中成功克隆了NAP基因,并获得重组蛋白的表达,为NAP幽门螺杆菌疫苗研制奠定了良好的基础.

Cloning, expression and sequencing of NAP of helicobacter pylori isolated from children

Objective： To amplify NAP gene from genome of Helicobacter pylori （HP） GZCH1 strain isolated from children, clone NAP gene into T vector, subclone into pGEX-4T-1 expression plasmid for gene sequencing and alignment, so as to provide basis for development of Helicobacter pylori vaccines. Methods： A pair of specific primers was designed according to Helicobacter pylori NAP gene in the GenBank. The full-length NAP gene of Helicobacter pylori strain isolated from children was amplified. The products were then ligated with pMD-T plasmid,and transformed to E. coli DH5a. The generated plasmid was verified by enzyme digestion and sequencing. After digested by EcoR I and Not I ,the pMD-T-NAP plasmid was ligated to corresponding pGEX- 4T-1. The recombinant plasmid was transformed into E. coli BL21. Expression of recombinant protein was induced by IPTG. Finally, gene sequencing and alignment were performed. Results： The 435 bp NAP gene was successfully amplified from Helicobacter pylori strain GZCH1 clinically isolated from children. The target gene fragment was identified by double enzyme digestion of recombinant pGEX-4T-1-NAP. DNA sequencing showed that NAP was in the desired reading frame, 99.2% consistent with Helicobacter pylori strain J99 by sequence alignment. After IPTG induction, fusion protein expression of pGEX-4T-1-NAP/BL21 was seen at about 42.8 kD. The sequence of NAP of Helicobacter pylori strain GZCH1 was submitted to GenBank （ accession number： GU301881 ）. Conclusions： NAP of Helicobacter pylori strain GZCH1 was successfully cloned with expression of recombinant protein,which provides a robust basis for development of Helicobacter pylori vaccines.