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目的构建幽门螺杆菌(Hp)热休克蛋白A(HspA)亚单位核酸疫苗,为进一步研制Hp核酸多价疫苗提供技术平台。方法以PMD-HspA为模板,扩增、纯化Hp hspA基因,插入到真核表达载体PcDNA3中,转化大肠埃希菌DH5a,经氨苄西林抗性筛选阳性克隆,得到重组核酸疫苗pcDNA3-HspA,用特异性PCR方法和小提质粒酶切电泳鉴定重组质粒,同时将pcDNA3-HspA进行测序,进一步确定是否构建成功及鉴定构建过程中是否发生基因突变。结果 PCR鉴定结果显示,扩增产物为0.36kb的目的基因;酶切鉴定结果显示,得到5.45kb克隆载体片段和0.36kb的目的基因片段;重组质粒pcDNA3-HspA测序,得到基因全长357bp目的基因片段,与克隆质粒测序图比较,无基因变异。结论成功构建了Hp单价核酸疫苗pcDNA3-HspA,为研制Hp核酸多价疫苗奠定基础。
Objective To construct HspA HspA subunit nucleic acid vaccine and provide a technological platform for the further development of Hp nucleic acid multivalent vaccine. Methods PMD-HspA was used as a template to amplify and purify Hp hspA gene. The Hp hspA gene was inserted into eukaryotic expression vector pcDNA3 and transformed into Escherichia coli DH5a. Positive clones were screened by ampicillin resistance to obtain recombinant DNA vaccine pcDNA3-HspA. Specific PCR and small plasmid restriction enzyme digestion were used to identify the recombinant plasmids. At the same time, pcDNA3-HspA was sequenced to determine whether the recombinant plasmids were successfully constructed and whether gene mutations occurred during the construction. Results PCR identification showed that the amplified product was 0.36kb. The restriction enzyme digestion showed that a 5.45kb cloned vector fragment and a 0.36kb gene fragment were obtained. The recombinant plasmid pcDNA3-HspA was sequenced and the full-length 357bp gene was obtained Fragment, compared with cloned plasmid sequencing map, no gene mutation. Conclusion The Hp monovalent nucleic acid vaccine pcDNA3-HspA was successfully constructed, which laid the foundation for the development of Hp nucleic acid multivalent vaccine.