结核杆菌HSP65与IL-12多价DNA疫苗的构建

Construction of the Multivalent DNA Vaccine of Mycobacterium Tuberculosis HSP65 and Cytokine IL-12 and its Identification

〔目的〕构建结核杆菌H37RV株HSP65基因与IL-12基因的共表达载体pVIVO2-HSP65-IL-12,并研究其在Vero-E6细胞内表达的可行性,为新一代结核多价DNA疫苗寻找理论依据。〔方法〕采用PCR技术,从培养的结核杆菌H37RV中抽提HSP65基因,克隆到pVIVO2-MCS上的一个多克隆位点,构建pVIVO2-HSP65,将质粒pORF-IL-12上的IL-12基因经双酶切后,亚克隆到pVIVO2-HSP65上,构建成共表达载体pVIVO2-HSP65-IL-12。并经XbaI/AvrII和NcoI/NheI进行双酶切和测序鉴定;用间接免疫荧光法(IFA)检测HSP65和mIL-12基因在Vero-E6细胞中的表达。〔结果〕双酶切和测序分析证明HSP65基因和mIL-12基因成功克隆到pVIVO2-MCS上,且方向正确,序列无突变;间接免疫荧光试验结果为阳性。〔结论〕共表达载体pVIVO2-HSP65-IL-12构建成功,且pVIVO2-HSP65-IL-12可在Vero-E6细胞中获得共表达。

Objective To construct the recombinant plasmid eo-expressioned by heat shock protein 65 （HSP65） of mycobacterium tuberculosis and IL-12 to express this plasmid in vero-E6 cell for providing evidence of the development of a new muhivalent DNA vaccine for tuberculosis. Method HSP65 gene was amplified by PCR with specific primers from the genome of myeobacterium tuberculosis H37RV and IL-12 gene was digested from plasmid Porf-IL-12 with restriction endonucleases NcoI and NheI . The products were further inserted into the two multiple cloning sites in vector pVIVO2 for construction the plasmid pVIVO2-HSP65-IL- 12. The resulting construct was identified by restriction endonuclease digestion and sequence analysis. This recombinant plasmid was then transfected into vero-E6 cell by lipofectamine and its expression was analyzed by means of indirect fluorescence assay （IFA）. Result It was found that the constructed recombinant plasmid containing the sequences of HSP65 and IL-12 obtained after double enzymes digestion and sequence analysis. It was proved by IFA that the recombinant plasmid pVIVO2-HSP65-IL-12 could express in vero-E6 cell. Conclusion The recombinant plasmid pVIVO2-HSP65-IL-12 has been constructed successfully, The HSP65 gene and IL-12 gene can co-express in vero-E6 cell.