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目的构建携带小鼠IL-17a编码基因的重组耻垢分枝杆菌(Recombinant Mycobacterium smegmatis,rMs),并研究其对巨噬细胞表达炎症因子的影响。方法双酶切质粒pET28a/mIL-17a和pMFA41,转化感受态E.coli TOP10,构建大肠杆菌-分枝杆菌穿梭表达质粒pMFA41/mIL-17a,电转化耻垢分枝杆菌mc2155,经SDS-PAGE和Western blot鉴定表达产物。分别以MOI为10∶1加入rMs、rMs+anti-IL-17A、Ms感染小鼠巨噬细胞系RAW264.7,并设PBS对照组。荧光定量PCR法检测转染细胞中β防御素-2(Defensinβ2,Defb-2)、巨噬细胞炎症蛋白(Macrophage inflammatory protein,MIP)-1α和MIP-2β基因mRNA水平;ELISA法检测细胞培养上清中MIP-1α、MIP-2β、IL-4及IFNγ蛋白的表达水平。结果重组表达质粒pMFA41/mIL-17a经双酶切及测序鉴定证明构建正确;表达的重组蛋白mIL-17A可与大鼠抗小鼠IL-17A单克隆抗体特异性结合,在相对分子质量约20 000处可见特异性反应条带;rMs组RAW264.7细胞中Defb-2、MIP-1α、MIP-2β基因mRNA水平较Ms组及PBS组明显升高(P<0.01);rMs组细胞培养上清中MIP-1α、MIP-2β、IL-4和IFNγ的蛋白浓度较Ms组及PBS组明显升高(P<0.01)。结论成功构建了表达具有生物学活性mIL-17A的重组耻垢分枝杆菌疫苗,该疫苗可有效促进体外培养的巨噬细胞表达MIP、Defb2、IFN-γ及IL-4,为新型疫苗的研发奠定基础。
Objective To construct Recombinant Mycobacterium smegmatis (rMs) carrying mouse IL-17a gene and study its effect on the expression of inflammatory cytokines in macrophages. Methods Plasmid pET28a / mIL-17a and pMFA41 were double digested and transformed into competent E. coli TOP10 to construct E. coli-mycobacterium shuttle plasmid pMFA41 / mIL-17a. The mycobacterium smegmatis mc2155 was transformed into E.coli by SDS-PAGE And Western blot to identify the expression product. The murine macrophage cell line RAW264.7 was infected with rMs + anti-IL-17A, Ms at an MOI of 10: 1, respectively, and PBS control group was established. The mRNA levels of Defensinβ2, MIP-1α and MIP-2β in transfected cells were detected by real-time fluorescence quantitative PCR. The expression of MIP- The expression levels of MIP-1α, MIP-2β, IL-4 and IFNγ in Chingqing were significantly higher than those in control group. Results The recombinant plasmid pMFA41 / mIL-17a was confirmed by double enzyme digestion and sequencing. The recombinant protein mIL-17A was specifically expressed in mouse anti-mouse IL-17A monoclonal antibody. (P <0.01). The expression of mRNA of Defb-2, MIP-1α and MIP-2β in RAW264.7 cells in rMs group was significantly higher than that in Ms and PBS groups (P <0.01) The protein concentrations of MIP-1α, MIP-2β, IL-4 and IFNγ were significantly higher than those in Ms and PBS groups (P <0.01). Conclusion The recombinant Mycobacterium smegmatis vaccine expressing mIL-17A has been successfully constructed, which can effectively promote the expression of MIP, Defb2, IFN-γ and IL-4 in macrophages cultured in vitro. Lay the foundation.