IFN-γ与HIV-1gp120融合蛋白的表达及其免疫调节作用

目的：研究gamma干扰素（IFN-γ）对人类免疫缺陷病毒（HIV-1）gp120蛋白免疫小鼠后效果的调节作用。方法：利用分子生物学技术，构建表达中国流行株HIV-1gp120N端片段基因的原核质粒pet44b-gp120，及共表达HIV-1gp120N端片段基因与IFN-γ基因的原核质粒pet44b-gp120／IFN-γ，在E．coli BL21（DE3）细胞中进行低温诱导蛋白表达，然后经纯化后免疫BALB／c小鼠。实验设皮下注射gp120／IFN-γ组、gp120组和PBS三组，以检测HIV-1gp120诱导的T细胞增殖能力，CTL杀伤作用以及Th1型细胞因子（IL-2，IFN-γ）和Th2型细胞因子（IL-4，IL-10）的表达情况。结果：通过PAGE和Western blot检测，证实上述两种蛋白在DE3细胞中得到表达。免疫学实验表明，针对HIV-1gp120的特异性T淋巴细胞增殖作用、特异性CTL杀伤作用，以及Th1型细胞因子IL-2和IFN-γ表达在三组之间均有显著性差异（P〈0．05），gp120／IFN-γ组高于gp120组，gp120组高于PBS组（P〈0．05）。反映体液免疫反应的Th2型细胞因子IL-4和IL-10的表达在三组之间没有显著性差异（P〉0．05）。结论：协同应用IFN-γ基因可明显加强HIV-1gp120基因的细胞免疫反应。

Expression of recombinant plasmid of HIV-1 gp120 and IFN-γ in E. coli and its immunoregulatory effect

OBJECTIVE: To investigate the regulating effect of interferon-gamma (IFN-gamma) gene on the immune response induced by human immunodeficiency virus (HIV) gp120 in mice. METHODS: The recombinant prokaryotic plasmid pet44b with HIV-1gp120 N and pet44b with HIV-1gp120 N linking IFN-gamma were constructed and expressed in E. coli BL21 (DE3) through IPTG. The purified proteins gp120 N and gp120 N/IFN-gamma were used in the following experiments: mice were injected with PBS i. s as control group 1, injected i. s with gp120 N as control group 2, and injected i. s with gp120 N/IFN-gamma as experiment group.The serum samples and spleen cells were collected 2 weeks after completion of immune program.T cells enlargement stimulated by gp120, CTL test and the detection of Th1 cytokines (SKs) IL-2, IFN-gamma and Th2 CKs IL-4, IL-10 were performed. RESULT: SDS-PAGE and Western blot confirmed that gp120 N and gp120 N/IFN-gamma fusion proteins were successfully expressed. The immune experiments showed that the enlargement of specific T lymphocytes, the response of CTL and the expression of Th1 type CKs IL-2 and IL-gamma against gp120 were significantly different among the three groups (P < 0.05). And the response of gp120 N/IFN-gamma group was stronger than that of gp120 N group, the latter was stronger than that of PBS group (P < 0.05). The differences in expression of Th2 type CKs IL-4 and IL-10 were not significant among the three groups (P >0.05). CONCLUSION: The cell specific immune responses of BALB/c mice to HIV-1CNgp120 antigen can be enhanced when HIV-1CNgp120 gene and IFN-gamma gene are used in coordination.