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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EcoRⅠ、NotⅠ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,pGEX-4T-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GZCHl 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。
OBJECTIVE: To amplify the neutrophil activating protein (NAP) gene from the genome of Helicobacter pylori isolates of GZCH1 and cloned into T vector and subcloned into expression vector pGEX-4T-1 for sequencing and gene alignment. Helicobacter pylori vaccine laid the foundation for the development. Methods: According to the NAP sequence of Helicobacter pylori in GenBank, a pair of specific primers was designed to amplify the full-length NAP gene of Helicobacter pylori clinical isolates. The full length NAP gene was ligated with T vector and transformed into E. coli DH5α. The plasmid was digested and sequenced. After digested with EcoRⅠ and NotⅠ, the recombinant plasmid pGEX-4T-1 was ligated with pGEX-4T-1. The recombinant plasmid was transformed into E. coli BL21. The plasmid was digested with restriction endonuclease and identified by IPTG. The recombinant protein was induced by IPTG. Results: The NAP gene was successfully amplified from the Helicobacter pylori isolates GZCH1. The size of the gene was 435 bp. The recombinant plasmid pGEX-4T-1-NAP was identified by restriction enzyme digestion. The sequencing results showed that the NAP gene was correctly read Sequence analysis showed 99.2% amino acid identity with Helicobacter pylori strain J99. After induced by IPTG, the expression of pGEX-4T-1-NAP / BL21 fusion protein was observed at the corresponding molecular weight of 42.8 kD. The Helicobacter pylori isolates GZCH1 NAP have been registered in GenBank (Accession number: GU301881). Conclusion: NAP gene was successfully cloned from Helicobacter pylori clinical isolates of GZCH1 and the recombinant protein was expressed, which laid a good foundation for the development of NAP Helicobacter pylori vaccine.