结核分枝杆菌CFP10-ESAT6融合蛋白免疫小鼠效果初步评价  

Preliminary evaluation of immunization effect of Mycobacterium tuberculosis fusion protein CFP10-ESAT6 in mice

作　　者：栾秀丽 范雪亭 王瑞欢 李马超[1] 于晋杰 钱程宇 曹斌 赵秀芹[1] 刘志广[1] 刘海灿[1] 李桂莲[1] 万康林[1] LUAN Xiu-li;FAN Xue-ting;WANG Rui-huan;LI Ma-chao;YU Jin-jie;QIAN Cheng-yu;CAO Bin;ZHAO Xiu-qin;LIU Zhi-guang;LIU Hai-can;LI Gui-lian;WAN Kang-lin(State Key Laboratory for Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;School of Public Health,University of South China,Hengyang 421001,China;College of Laboratory Medicine and Life Sciences,Wenzhou Medical University,Wenzhou 325035,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206 [2]南华大学公共卫生学院,衡阳421001 [3]温州医科大学检验医学院与生命科学学院,温州325035

出　　处：《中国人兽共患病学报》2022年第7期589-596,共8页Chinese Journal of Zoonoses

基　　金：“一带一路”重要传染病协同防控关键技术研究(No.2018ZX1010-1002-004)。

摘　　要：目的融合表达和纯化结核分枝杆菌(MTB)CFP10-ESAT6(CE),初步评价免疫效果。方法构建pET32a(+)-CE重组质粒,E.coli BL21(DE3)中融合表达,Ni-NTA层析纯化。用CE加铝佐剂,50μg CE/只/次,分3次免疫BALB/c小鼠。ELISA测定血清IgG抗体;ELISpot与Luminex检测细胞因子,进行MTB体外生长抑制试验。卡介苗(BCG)为阳性对照。结果CE免疫小鼠,能诱导产生高效价的IgG、IgG1和IgG2a,且IgG1(t=19.1,P<0.0001)和IgG2a(t=8.7,P<0.0001)抗体水平均高于BCG组。CE组与BCG组诱导产生的分泌IFN-γ的斑点形成细胞(SFC)数量差异无统计学意义(t=0.4,P>0.05),但诱导分泌IL-4的SFC数量高于BCG组(t=8.0,P<0.0001)。CE组诱导分泌GM-CSF(t=8.4,P<0.05)、IL-6(t=8.3,P<0.05)、IL-10(t=2.5,P<0.05)和IL-4(t=7.0,P<0.05)均高于BCG组,而CE组诱导分泌IFN-γ(t=1.4,P>0.05)、TNF-α(t=1.8,P>0.05)、IL-2(t=2.0,P>0.05)、IL-12(t=0.9,P>0.05)和IL-17(t=1.3,P>0.05)均与BCG组相似,差异无统计学意义。CE组小鼠脾细胞的MTB体外生长抑制结果与BCG组相似(t=0.8,P>0.05)。结论CE免疫小鼠可诱导较强的Th1与Th2混合型免疫反应,且有较强的体外抑制MTB生长的能力,表明CE具有良好的结核疫苗研制应用价值。The purpose of this study was to construct and purify a fusion expression and purification of the protein CFP10-ESAT6(CE)of Mycobacterium tuberculosis(MTB)and then preliminarily evaluated its immune effect.The recombinant plasmid pET32a(+)-CE was constructed,with which CE was expressed in E.coli BL21(DE3)and purified by Ni-NTA chromatography.BALB/c mice were immunized with CE adjuvanted with aluminum for three times,the dosage of CE was 50μg per mouse per time.Serum IgG antibodies were determined by ELISA;cytokines were detected by ELISpot and Luminex;In vitro growth inhibition test of MTB was performed.BCG was used as the positive control.CE fusion protein immunized group induced high expression of IgG,IgG1 and IgG2a,and the levels of IgG1(t=19.1,P<0.0001)and IgG2a(t=8.7,P<0.0001)antibodies in group CE were significantly higher than those in group BCG.No statistical difference was found in the number of IFN-γ-secreting spot-forming cells(SFC)induced by the CE and BCG groups(t=0.4,P>0.05),while the number of IL-4-secreting SFCs induced by group CE was significantly higher than that by group BCG(t=8.0,P<0.0001).The levels of GM-CSF(t=8.4,P<0.05),IL-6(t=8.3,P<0.05),IL-10(t=2.5,P<0.05)and IL-4(t=7.0,P<0.05)induced by the CE group were significantly higher than those induced by the BCG group,however,no statistically significant difference was found in the levels of IFN-γ(t=1.4,P>0.05),TNF-α(t=1.8,P>0.05),IL-2(t=2.0,P>0.05)、IL-12(t=0.9,P>0.05)和IL-17(t=1.3,P>0.05)induced by these two groups.In vitro growth inhibition of MTB with spleen cells of the mice in the CE group was similar to that in the BCG group(t=0.8,P>0.05).Mice immunized with CE can induce a strong Th1/Th2-mixed-type immune response,and had a strong ability to inhibit the growth of MTB in vitro,indicating that CE has a good value in the development and application of new tuberculosis vaccine.

关 键 词：结核分枝杆菌 培养滤液蛋白10 早期分泌抗原靶6 重组融合蛋白CE 疫苗 

分 类 号：R378.91[医药卫生—病原生物学]