结核分枝杆菌TB10.4亚单位疫苗的构建及免疫原性研究

Construction and Immunogenicity of Mycobacterium Tuberculosis Subunit Vaccine TB10.4

目的构建结核分枝杆菌TB10.4基因的原核表达载体,表达和纯化该蛋白,构建亚单位疫苗,并对其免疫原性进行研究。方法从重组质粒p ET-SUMO-TB 10.4获得基因片断后,将其插入到载体p ET-28a(+),转化Ecoli DH5α感受态细胞,随机筛选阳性克隆,测序正确后并将该重组质粒转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,经SDS-PAGE分析后,用Ni-NTA亲和层析柱进行纯化并复性。随机将C57BL/6雌性小鼠分为Tris-HCl组、TB10.4组(佐剂为DDA/Poly I∶C)及卡介苗(BCG)3组,每组10只,每只小鼠每次免疫200μL,其中DDA250μg及Poly I∶C 25μg,蛋白量为20μg,而BCG为5×106CFU,分别于1、4、7周皮下免疫小鼠。末次免疫4周后,用特异性抗原TB10.4刺激脾淋巴细胞,检测其分泌IFN-γ水平;用ELISA检测血清特异性抗体Ig G2b、Ig G1。结果构建了原核表达重组质粒p ET-28a-TB10.4,并在分子量约为10.4k Da处观察到蛋白条带;小鼠血清中产生特异性抗体Ig G2b与Ig G1,体外脾淋巴细胞受TB10.4刺激时分泌IFN-γ水平显著高于Tris-HCl组(P<0.05)及BCG组(P<0.05)。结论 TB10.4蛋白与佐剂构建了亚单位疫苗,该疫苗可在C57BL/7小鼠中诱导抗原特异性免疫应答,可作为新型结核亚单位疫苗的候选疫苗予以进一步研究。

Objective To construct prokaryotic expression vector consisted of TB10. 4,and to express and purify the protein,then to investigate its immunogenicity. Methods The gene was obtained from p ET- SUMO- TB10. 4,then inserted into p ET- 28 a. The recombinant plasmid was transformed into E. coli DH5α while positive clones were randomly selected,then the plasmid was transformed into competent E. coli BL21( DE3)after sequenced correctly. Recombinant protein was induced by IPTG and identified by SDS- PAGE analysis,and then the protein was purified by Ni- NTA resin. Female C57 BL /6 mice were randomly divided into three groups called Trs- HCl + DDA / Poly I∶ C,TB10. 4 + DDA / Poly I∶ C and BCG,ten mice per group. The mice were immunized subcutaneously at weeks 1,4,7. The level of IFN- γ secreted by spleen lymphocytes stimulated by TB10. 4 was detected at the last immunization. The antibodies of Ig G2 b and Ig G1 in sera were detected by Elisa. Results Recombinant plasmid pet28a- TB10. 4 was constructed,and the protein band about10. 4 Kda was observed. The mice immunized by TB10. 4 could induce antigen- specific antibody of Ig G2 b and Ig G1 in sera. The IFN- γ level in TB10. 4 group was significantly higher than that of Trs- HCl and BCG group.Conclusion The subunit vaccine was constructed by protein TB10. 4 and adjuvants. It can induce antigen-specific immune response. It can be used as a candidate of novel TB subunit vaccine for further evaluation.