结核分枝杆菌三基因融合表达的亚单位疫苗研究

Study on subunit vaccines of three genes fusion expression of Mycobacterium tuberculosis

为筛选出安全、有效的抗结核分枝杆菌感染的亚单位疫苗,本研究选择了9个结核分枝杆菌的重要抗原(Rv0199、Rv0394c、Omp A、Rv3802c、Ag85a、TB10.4、Rv2660c、Rv0125、Mpt51),利用小鼠模型评价其免疫效果。每3个抗原一组串联融合表达,将其分别与弗氏不完全佐剂及沙门氏菌菌蜕混合,共分成7组,通过皮下注射的方式免疫小鼠,共免疫3次。每次免疫后两周检测其血清抗体分型与效价、脾脏淋巴细胞的分化及其细胞因子的分泌。结果表明,这些融合蛋白均能够刺激机体产生高滴度的抗体,具有较强的免疫原性,其中融合蛋白Rv0199-Rv0394c-Omp A在一免后2周即可以刺激机体产生较高滴度的抗体。抗体分型结果显示,除阴性对照组外,其余组小鼠血清中Ig G1的滴度均高于Ig G2a。并且机体的免疫反应倾向于体液免疫。其中,沙门氏菌菌蜕的佐剂作用与商品化的弗氏不完全佐剂免疫效果一致。本研究为研制结核分枝杆菌亚单位疫苗奠定了基础。

To screen safe and effective subunit vaccines against Mycobacterium tuberculosis infection, nine important antigens （Rv0199, Rv0394c, OmpA, Rv3802c, Ag85a, TB10.4, Rv2660c, Rv0125, Mpt51） of Mycobacterium tuberculosis were selected and the effect of the immunization was evaluated by mouse model. The fusion proteins were expressed in the series connection of every three antigens. The mice were immunized three times with the fusion proteins mixed with incomplete freund＇s adjuvant or Salmonella ghost by subcutaneous injection. The titer and differentiation of the antibody in mice were detected two weeks after each immunization. In parallel, the differentiation of spleen lymphocytes cell and the cytokines were tested. The results showed that these fusion proteins, with strong immunogenicity, could stimulate the body to produce high titer of antibodies. The fusion protein Rv0199-Rv0394c-OmpA could stimulate the body to produce high titer of antibodies only 2 weeks after prime immunization. In all groups, except the control, the titers of IgG1 of serum in mice were higher than that of IgG2a, which indicated that the body＇s immune reaction tended to humoral immunity. Among them, the role of the ghost of Salmonella as an adjuvant was competitive with that of commercial incomplete freund＇s adjuvant.