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目的:构建克隆结核分枝杆菌Mtb8.4/hIL12嵌合基因疫苗,并在COS7细胞中表达,研究该疫苗的免疫原性。方法:克隆Mtb8.4/hIL12嵌合基因,并导入真核表达载体pCIneo中,构建pCIneoMtb8.4/hIL12重组真核质粒。用限制性内切酶消化、PCR及DNA序列测定等鉴定后,转染COS7细胞,用RTPCR和Westernblot鉴定Mtb8.4/hIL12嵌合基因在转录水平的表达。用Mtb8.4/hIL12嵌合基因疫苗免疫C57BL/6N小鼠,收集脾细胞培养上清检测细胞因子的水平,并用乳酸脱氢酶(LDH)释放法测定细胞毒性T细胞(CTL)的杀伤作用。结果:pCIneoMtb8.4/hIL12重组质粒构建成功。以其转染COS7细胞后,Mtb8.4/hIL12嵌合基因在转录水平获得表达。Mtb8.4/hIL12嵌合基因疫苗能诱导较强的抗原特异性Th1型细胞免疫应答,IFNγ和IL2的分泌增加,IL4的分泌减少,特异性CTL的杀伤活性增强。结论:成功地构建pCIneoMtb8.4/hIL12重组质粒,并在COS7细胞中表达。构建的Mtb8.4/hIL12基因疫苗具有较强的免疫原性,可明显诱导CTL的杀伤活性。
OBJECTIVE: To construct a chimeric gene vaccine against Mycobacterium tuberculosis Mtb8.4 / hIL12 and express it in COS7 cells to study the immunogenicity of the vaccine. Methods: The Mtb8.4 / hIL12 chimeric gene was cloned and inserted into eukaryotic expression vector pCIneo to construct pCIneoMtb8.4 / hIL12 recombinant eukaryotic plasmid. After restriction endonuclease digestion, PCR and DNA sequencing, the recombinant plasmid was transfected into COS7 cells and the expression of Mtb8.4 / hIL12 chimeric gene was identified by RTPCR and Western blot. C57BL / 6N mice were immunized with Mtb8.4 / hIL12 chimeric gene vaccine and the levels of cytokines in spleen cell culture supernatants were collected. The killing effect of cytotoxic T lymphocytes (CTLs) was determined by lactate dehydrogenase (LDH) release assay . Results: pCIneoMtb8.4 / hIL12 recombinant plasmid was successfully constructed. After transfected into COS7 cells, Mtb8.4 / hIL12 chimeric gene was expressed at the transcriptional level. Mtb8.4 / hIL12 chimeric gene vaccine can induce a strong antigen-specific Th1-type cellular immune response, increased secretion of IFNγ and IL2, decreased secretion of IL4 and enhanced cytotoxic activity of specific CTLs. Conclusion: The pCIneoMtb8.4 / hIL12 recombinant plasmid was successfully constructed and expressed in COS7 cells. The constructed Mtb8.4 / hIL12 gene vaccine has strong immunogenicity and can obviously induce CTL killing activity.