幽门螺杆菌热休克蛋白A核酸疫苗的构建

Construction of Helicobacter pyloriHspA nucleic acid vaccines

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 Helicobacter pylori（Hp） heat shock protein A（HspA） subunit nucleic acid vaccines so as to provide the technology platform for further study of the multivalent nucleic vaccine of Hp.METHODS The Hp HspA gene was amplified from pMD-HspA plasmid DNA by PCR and was inserted into the vector pcDNA3,and then were transformed into the DH5a of Escherichia coli strain.The recombinant plasmid was detected by ampicillin antibiotic and was identified by digesting recombinant plasmid with restriction endonucleases,specific PCR and determining nucleotide sequence.RESULTS Specific PCR could amplify 0.36kb HspA gene fragment.At the same time,the recombinant plasmid of HspA was digested into two fragments,which were eucaryotic expression vector fragment（5.45kb） and HspA gene fragment（0.36kb）.Gene sequencing result showed that HspA gene fragment was 357bp long.It was the same as the first sequencing result.The Hp HspA gene was inserted correctly to the target site of pcDNA3.0,and no gene mutation was detected.CONCLUSION Hp HspA univalent DNA vaccine pcDNA3-HspA is constructed successfully,which makes an experimental foundation for the development of the Hp multivalent nucleic acid vaccine.