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目的用分子生物学方法制备H37Rv的两种抗原Ag85b、HspX,通过与铝佐剂和CpG佐剂联用免疫小鼠进行初步药理学实验以观察其生物学活性。方法常规方法分别构建重组表达质粒pET30a-Ag85b和pET30a-HspX,内切酶鉴定目的片段后两种质粒分别转化BL-21大肠杆菌,经异丙基-β-D-硫代半乳糖苷诱导后分别产生两种蛋白;用阴离子交换柱Source30、QHP以及疏水层析柱对两种蛋白进行纯化;蛋白测序进行鉴定。纯化后的两种蛋白分别与铝佐剂和CpG佐剂联用(Ag85b,Ag85b+Al,Ag85b+CpG,Ag85b+Al+CpG;HspX,HspX+Al,HspX+CpG,HspX+Al+CpG),免疫C57/BL小鼠,同时设置生理盐水对照组,取脾细胞进行酶联免疫斑点实验(ELISPOT)检测干扰素-γ(IFN-γ)的分泌;同时进行淋巴细胞增殖实验检测脾淋巴细胞经体外刺激后的增殖情况。结果成功构建了两种重组表达质粒pET30a-Ag85b和pET30a-HspX,成功诱导表达了两种蛋白;经过纯化,两种蛋白纯度均达到95%;经鉴定两种蛋白纯化产物的N端15个氨基酸序列与目的序列相同;Ag85b+CpG、Ag85b+Al和Ag85b+CpG+Al组经Ag85b(80μg/ml)体外刺激后分泌IFN-γ显著升高,与生理盐水组比较差异具有显著性(P<0.05);HspX+CpG+Al组经HspX(80μg/ml)体外刺激后分泌IFN-γ显著升高,与生理盐水组比较差异具有显著性(P<0.05)。结论成功表达并纯化了结核分枝杆菌H37Rv的两种抗原Ag85b和HspX,与铝佐剂和CpG佐剂联用后成功刺激小鼠产生了细胞免疫反应,证实了两种重组蛋白的生物学活性,为下一步药效学评估奠定了基础。
OBJECTIVE To prepare two antigens of Ag85b and HspX of H37Rv by molecular biology method and conduct preliminary pharmacological experiments to observe their biological activity by immunizing mice with aluminum adjuvant and CpG adjuvant. Methods The recombinant plasmids pET30a-Ag85b and pET30a-HspX were constructed by conventional methods. After the target fragment was identified by restriction endonuclease, the two plasmids were respectively transformed into BL-21 E.coli. After induced by isopropyl-β-D-thiogalactoside Two kinds of proteins were produced respectively. Two kinds of proteins were purified by anion exchange column Source30, QHP and hydrophobic column. The protein was identified by sequencing. The purified two proteins were respectively combined with aluminum adjuvant and CpG adjuvant (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG) , And immunized C57 / BL mice. At the same time, normal saline control group was set up. Spleen cells were harvested for enzyme-linked immunospot assay (ELISPOT) to detect the secretion of interferon-γ (IFN-γ). Lymphocyte proliferation assay was also performed to detect spleen lymphocytes Proliferation after in vitro stimulation. Results Two recombinant plasmids, pET30a-Ag85b and pET30a-HspX, were successfully constructed and successfully expressed. After purification, the purity of the two proteins reached 95%. The N-terminal 15 amino acids of the two purified proteins were identified The results showed that IFN-γ secreted by Ag85b + CpG, Ag85b + Al and Ag85b + CpG + Al groups was significantly increased after stimulated with Ag85b (80μg / ml) in vitro. The difference was significant compared with saline group 0.05). The secretion of IFN-γin HspX + CpG + Al group stimulated by HspX (80μg / ml) in vitro was significantly higher than that of saline group (P <0.05). Conclusions The two antigens Ag85b and HspX of Mycobacterium tuberculosis H37Rv were successfully expressed and purified. The combination of aluminum adjuvant and CpG adjuvant successfully stimulated the mice to produce cellular immune response, and confirmed the biological activity of the two recombinant proteins , Which laid the foundation for the next pharmacodynamic evaluation.