嗜肺军团菌热休克蛋白60基因真核表达载体的构建及鉴定

Construction and Identification of Legionella pneumophila Heat Shock Protein 60 Gene Eukaryotic Expression Vector

目的构建嗜肺军团菌热休克蛋白60(HSP60)基因的真核表达载体pcDNA3.1/HSP60,并对其进行鉴定。方法应用聚合酶链式反应(PCR)技术从嗜肺军团菌扩增得到HSP60基因,并将其克隆至载体pUC18进行序列测定。将重组质粒pUC18/LpHSP60中的目的基因亚克隆入真核表达载体pcDNA3.1(+),构建重组子pcDNA3.1/HSP60,通过限制性内切酶酶切及PCR进行鉴定。结果克隆的LpHSP60基因序列与GenBank公布的一致性为98%,限制性内切酶酶切及PCR分析表明HSP60基因已成功插入pcDNA3.1中。结论成功构建了LpHSP60基因的真核表达载体,为进一步研究军团菌病HSP60基因DNA疫苗奠定了基础。

Objective To construct and identify the eukaryotic expression vector of the heat shock protein 60 （HSP60） gene of Legionella pneumophila. Methods The HSP60 gene of Legionella pneumophila was amplified by PCR and then cloned into vector pUC18 for sequencing. The target gene in the recombinant plasmid （ pUC18/Hsp60 ） was sub - cloned into the eukaryotic expression vector pcDNA3.1 （ ＋ ） and the recombinant pcDNA3. 1/HSP60 was constructed. This recombinant plasmid was identified by restriction analysis and PCR. Results Comparison of the sequence of HSP60 gene with that reported in GenBank showed that the identities were 98%. And it was comfirmed that HSP60 gene was successfully inserted into pcDNA3.1. Conclusion We successfully constructed the eukaryotic expression vector pcDNA3. 1/HSP60 and it laids a good foundation of the research of new legionellosis vaccine.