结核分枝杆菌Rv3621c原核表达及免疫功能研究

Prokaryotic expression and immune function in Mycobacterium tuberculosis Rv3621c

目的以结核分枝杆菌(Mycobacterium tuberculosis,M.tb)H37Rv基因组为模板,构建、纯化及鉴定原核表达质粒pPROEX-Rv3621c,通过人群、小鼠试验进行免疫原性评价。方法构建重组质粒pPROEX-Rv3621c,并以全血干扰素释放分析技术(Whole-blood IFN-γrelease assay,WBIA)检测其能否被安徽省淮南市M.tb感染者T细胞特异性识别。rRv3621c混合佐剂MTM[母牛分枝杆菌(M.vaccae),人工合成海藻糖-6′6,二分枝菌酸(TDB),单磷酰脂质A(MPLA)]免疫小鼠后,检测血清中特异性抗体分泌水平、脾细胞中抗原特异性Th1型细胞因子分泌水平及肺脏细胞因子mRNA表达水平。结果成功构建重组质粒pPROEX-Rv3621c,并使之诱导表达、纯化和鉴定。在rRv3621c蛋白诱导下,活动性结核(Active tuberculosis,ATB)患者外周血淋巴细胞释放的IFN-γ水平明显较高(t=4.813,P<0.01),且ATB患者产生的IFN-γ水平高于潜伏性结核(Latent tuberculosis infection,LTBI)人群(t=4.442,P<0.01)。BCG+Rv3621c/MTM组小鼠产生的特异性抗体滴度水平明显高于Rv3621c/MTM组(P<0.01)和BCG组(P<0.01),Rv3621c/MTM组和BCG+Rv3621c/MTM组小鼠的IgG2a/IgG1比值大于1,明显高于MTM组和BCG组。BCG+Rv3621c/MTM组小鼠均分泌高水平IFN-γ、TNF-α和IL-2。Rv3621c/MTM组小鼠肺脏组织中IFN-γ、TNF-α及iNOS表达水平较高。结论M.tb感染者外周血T细胞可特异性识别rRv3621c蛋白,rRv3621c混合佐剂MTM可以诱导较强烈的抗原特异性Th1型免疫应答。

A prokaryotic expression plasmid pPROEX-Rv3621c was constructed,purified,and identified using the Mycobacterium tuberculosis(M.tb)H37Rv genome as a template.Human and mice tests were performed to evaluate immunogenicity.The recombinant plasmid pPROEX-Rv3621c was constructed and tested with a whole-blood IFN-γrelease assay(WBIA)to determine whether it could be specifically recognized by the T cells of M.tb-infected individuals in Huainan City,Anhui Province.The specific antibody secretion levels in serum,the antigen-specific Th1-type cytokine secretion levels in splenocytes,and the mRNA expression levels of cytokines in the lungs were measured after immunizing mice with rRv3621c mixed adjuvant MTM[M.vaccae,trehalose-6,6′-dibehenate(TDB),monophosphoryl lipid A(MPLA)].Data indicated that the recombinant plasmid pPROEX-Rv3621c was constructed,expressed,purified,and identified successfully.Under the induction of the rRv3621c protein,the levels of IFN-γreleased from peripheral blood lymphocytes of patients with active tuberculosis(ATB)was significantly higher(t=4.813,P<0.01),and the levels of IFN-γproduced by ATB patients were higher than those of latent tuberculosis infection(LTBI)(t=4.442,P<0.01).The BCG+Rv3621c/MTM group produced significantly higher titers of specific antibodies than the Rv3621c/MTM group(P<0.01)and the BCG group(P<0.01).The IgG2 a/IgG1 ratios of the Rv3621c/MTM and BCG+Rv3621c/MTM groups were greater than 1 and significantly higher than that of the MTM and BCG groups.The mice of the BCG+Rv3621c/MTM group all secreted high levels of IFN-γ,TNF-α,and IL-2.The expression levels of IFN-γ,TNF-α,and iNOS in the lungs of mice in the Rv3621c/MTM group were higher.In conclusion,human M.tb-infected peripheral blood T cells can specifically recognize rRv3621c protein,and rRv3621c mixed adjuvant MTM can induce a stronger antigen-specific Th1-type immune response.