摘要
目的构建结核分枝杆菌EspB及EspBN蛋白原核表达系统,表达并纯化重组蛋白,评价两种蛋白的免疫原性。方法构建pGEX-4T-1-EspB及pGEX-4T-1-EspBN重组质粒,转化大肠埃希菌,经ITPG诱导表达后纯化获得目的蛋白。分别取两种蛋白与等体积弗氏完全佐剂乳化后免疫BALB/c小鼠,在初次免疫后第14、28 d以相同剂量蛋白和弗氏不完全佐剂加强免疫。试验设PBS对照组。每鼠注射100μL PBS溶液与等体积的弗氏不完全佐剂,末次免疫后第14、28、42 d小鼠取眼球采血,分离血清,采用West ern blot法检测抗血清的特异性,ELISA法检测血清抗体效价及IgG亚型。分离免疫小鼠脾淋巴细胞,体外经相应抗原刺激后,CCK8法检测脾淋巴细胞增殖水平,ELISA法检测培养液上清中IL-4、INF-γ水平。结果成功构建了高效原核表达重组质粒pGEX-4T-1-EspB及pGEX-4T-1-EspBN。重组质粒转化DE3后经IPTG诱导,稳定表达EspB融合蛋白和EspBN融合蛋白。分别用两种蛋白免疫小鼠,制备的抗血清均具有特异性,抗EspB蛋白血清与抗EspBN血清的总IgG及各亚型IgG效价均高于PBS组(均P<0.05)。EspB组和EspBN组脾淋巴细胞增殖刺激指数均高于PBS组(q值分别为7.17和3.47,均P<0.05)。免疫小鼠淋巴细胞经抗原刺激后IL-4释放水平均呈降低趋势,INF-γ释放水平均呈升高趋势(均P<0.05)。结论结核分枝杆菌EspB蛋白及EspBN蛋白均具有较强的免疫原性,为进一步分析其动物免疫保护效应等研究奠定了基础。
Objective To construct the prokaryotic expression system of Mycobacterium tuberculosis EspB protein and EspBN protein,purify the recombinant proteins and evaluate the immunogenicity of the proteins.Methods The genomic DNA of EspB and EspBN were amplified by PCR and cloned into prokaryotic expression vector.The fusion proteins were induced by IPTG in E.coli BL21(DE3)strains,purified by GST labeled protein purification kit and excised the GST labeled proteins to obtain the target proteins.BALB/c mice were immunized with two kinds of protein mixed with equal volume of Freund's incomplete adjuvant respectively.The same dose of protein and Freund's incomplete adjuvant were used to enhance the immunity on the 14th and 28th day after the first immunization.The mice in control group were only injected with 100μL PBS solution and equal volume of Freund's incomplete adjuvant.On the 14th,28th and 42th day after the last immunization,the eyeball blood was collected to prepare the antiserum.The specificity of each antiserum was detected by Western Blot,and the titer and IgG subtype of each antiserum were detected by ELISA.The proliferation of splenic lymphocytes was detected by CCK8 method,and the levels of IL-4 and INF-γwere detected by ELISA.Results The recombinant plasmids pGEX-4T-1-EspB and pGEX-4T-1-EspBN were successfully constructed,induced by IPTG and stably expressed fusion proteins in E.coli.After immunizing mice with the two proteins,the antisera obtained were specific.The total IgG and subtype IgG titers of anti EspB protein serum and anti EspBN protein serum were higher than those in PBS group.On the 28th and 42nd day after the last immunization,the proliferation stimulation index of splenic lymphocytes in EspB group were higher than that in PBS group.(q values were 7.17 and 3.47 respectively,with both P0.05).On the 42nd day after the last immunization,the proliferation stimulation index of splenic lymphocytes in EspBN group were higher than that in PBS group(q=3.47,P0.05).On the 42nd day after the last immunization,the proliferation stimulation index of splenic lymphocytes in EspB group were higher than that in EspBN group(q=3.70,P0.05).The release levels of IL-4 in EspB group and EspBN group showed a decreasing trend,were higher than that in PBS group on the 14th day after the last immunization(q values were 10.80 and 4.34 respectively,with both P0.05),and were lower than that in PBS group on the 42th day after the last immunization(q values were 7.08 and 6.12 respectively,with both P0.05).The release level of INF-γin EspB group and EspBN group showed an increasing trend,and were higher than that in PBS group on the 42th day after the last immunization(q values were 5.37 and 3.61 respectively,with both P<0.05).Conclusion The results showed that both EspB protein and EspBN protein could induce humoral and cellular immune responses in mice.
作者
戴晟晖
黄丹丹
DAI Shenghui;HUANG Dandan(School of Basic Medical Sciences and Forensic Medicine,North Sichuan Medical College,Nanchong 637000,Sichuan,China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2023年第6期677-682,共6页
Journal of Pathogen Biology
基金
四川省教育厅自然科学重点项目(No.13ZA0217)。
