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〔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 co-expression vector pVIVO2-HSP65-IL-12 of HSP65 gene and IL-12 gene of Mycobacterium tuberculosis H37RV strain and study the feasibility of its expression in Vero-E6 cells to find a new generation of multivalent DNA vaccine against tuberculosis Theoretical basis. [Methods] The HSP65 gene was extracted from the cultured Mycobacterium tuberculosis H37RV by PCR and cloned into a multiple cloning site on pVIVO2-MCS to construct pVIVO2-HSP65. The IL-12 gene on plasmid pORF-IL-12 After double digestion, it was subcloned into pVIVO2-HSP65 to construct a co-expression vector pVIVO2-HSP65-IL-12. Digested by XbaI / AvrII and NcoI / NheI. The expression of HSP65 and mIL-12 gene in Vero-E6 cells was detected by indirect immunofluorescence assay (IFA). [Results] Double enzyme digestion and sequencing analysis confirmed that HSP65 gene and mIL-12 gene were successfully cloned into pVIVO2-MCS with correct orientation and no mutation in the sequence. The result of indirect immunofluorescence assay was positive. [Conclusion] The co-expression vector pVIVO2-HSP65-IL-12 was successfully constructed, and pVIVO2-HSP65-IL-12 was co-expressed in Vero-E6 cells.