重组IL-17A耻垢分枝杆菌的构建及其对巨噬细胞表达炎症因子的影响

Construction of recombinant Mycobacterium smegmatis with IL-17A and Its effect on expression of inflammatory factor in macrophages

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 a recombinant Mycobacterium smegmatis（rMs） with mouse IL-17A gene and analyze its effect on expression of inflammatory factors in macrophages.Methods Plasmids pET28a / mIL-17a and pMFA41 were digested with BamHⅠ and Hind Ⅲ separately,and the target gene and vector fragments were recovered and linked,than transformed to E.coli Top10.The constructed E.coli-M.smegmatis shuttle expression plasmid pMFA41 / mIL-17a was transformed to M.smegmatis mc2 155 by electroporation,and the expressed product was identified by SDS-PAGE and Western blot.Mouse macrophages RAW264.7 were transfected with rMs,rMs ＋ anti-IL-17A and Ms each at a MOI of 10︰1,using PBS as control.The defensin β2（Defb-2）,macrophage inflammatory protein（MIP）-1α and MIP-2β levels in transfected RAW264.7 cells were determined by fluorescent quantitative PCR,while the expression levels of MIP-1α,MIP-2β,IL-4 and IFNγ in culture supernatant of the cells by ELISA.Results Restriction analysis and sequencing proved that recombinant plasmid pMFA41 / IL-17a was constructed correctly.The expressed recombinant mIL-17A showed specific binding to rat anti-mouse IL-17A monoclonal antibody,forming a reaction band with relative molecular mass of about 20 000.The Defb-2,MIP-1α,MIP-2β mRNA levels in RAW264.7 cells transfected with rMs,as well as the expression levels of MIP-1α,MIP-2β,IL-4 and IFNγ in culture supernatant of the cells,were significantly higher than those with Ms and PBS（P 0.01）.Conclusion Recombinant M.smegmatis express bioactive mIL-17A was successfully constructed,which promoted the expressions of MIP,Defb2,IFNγ and IL-4 in macrophages cultured in vitro.It laid a foundation of development of novel vaccines.