结核分枝杆菌新型抗原Rv3117联合DDA/MPL佐剂作为亚单位疫苗在小鼠体内的免疫评估

Immunological evaluation of subunit vaccine prepared by combining specific antigen Rv3117 of Mycobacterium tuberculosis with DDA/MPL adjuvant

目的用结核分枝杆菌(M.tuberculosis)特异性抗原Rv3117联合双十八烷基二甲基溴化铵(DDA)/单磷酰脂质体A(MPL)佐剂构建结核亚单位疫苗(Rv3117/DDA/MPL),并在C57BL/6小鼠体内评估其加强卡介苗(BCG)首次免疫后的免疫效应。方法采用分子克隆方法构建重组质粒p ET32a-Rv3117,转入感受态大肠埃希菌BL21(DE3)PLys S,诱导表达并纯化目的蛋白,用Triton X-114相分离法去除目的蛋白中的内毒素,然后与DDA/MPL佐剂充分乳化构建亚单位疫苗。C57BL/6小鼠随机分为对照组(未予任何处理)、PBS组(免疫PBS)、BCG组(单纯首次免疫BCG)、BCG+DDA/MPL组(首次免疫BCG,2周后用佐剂DDA/MPL加强免疫2次)和BCG+Rv3117/DDA/MPL组(首次免疫BCG后用亚单位疫苗Rv3117加强免疫2次),每组5只。分别采用酶联免疫斑点检测(ELISPOT)和酶联免疫吸附测定(ELISA)技术对免疫小鼠的细胞免疫和体液免疫进行评估。结果成功克隆表达并纯化结核特异性抗原Rv3117。ELISPOT和ELISA结果显示:受10.0μg/m L结核菌素纯化衍生蛋白(PPD)刺激后,与对照组、PBS组、BCG组和BCG+DDA/MPL组比较,BCG+Rv3117/DDA/MPL组小鼠T细胞产生γ干扰素(IFN-γ)的水平明显升高(P<0.05);BCG+DDA/MPL组培养上清液中肿瘤坏死因子(TNF-α)、白介素6(IL-6)、白介素12(IL-12p70)细胞因子水平均显著高于对照组和PBS组(P<0.05),总抗体Ig G和Ig G1亚型水平亦较高;而BCG+Rv3117/DDA/MPL组上述指标水平均显著优于BCG+DDA/MPL组(P<0.05)。结论亚单位疫苗Rv3117/DDA/MPL在C57BL/6小鼠体内可增强BCG的细胞免疫和体液免疫效应。结核分枝杆菌特异性抗原Rv3117有望成为新的结核疫苗设计及结核诊断的候选抗原。

Objective To construct a TB subunit vaccine （ Rv3117 / DDA/MPL） by combining specific antigen Rv3117 of Mycobacterium tuberculosis （M. tuberculosis） with the adjuvant of dimethyl dioctadecyl ammoniumbromide （DDA） /monophosphoryl lipid A （MPL） and to evaluate the immune responses after the bacillus calmetteguerin （BCG）-Prime in C57BL/6 mice. Methods The molecular cloning method was adopted to construct a recombinant plasmid pET32a-Rv3117 and the specific recombinant Rv3117 protein of M. tuberculosis expressed in Escherichia coli BL21 （DE3） PLysS was obtained. Then the antigen Rv3117 was induced and purified. The endotoxin was removed by Triton X-114 phase separation method and the subunit vaccine was prepared by fully emulsifying M. tuberculosis specific antigen Rv3117 with DDA/MPL adjuvant. The C57BL/6 mice were randomly divided into the control group （without any treatment）, PBS group （immunized with PBS）, BCG group （only immunized with BCG）, BCG ＋ DDA/MPL group （ immunized with DDA/MPL adjuvant twice after being immunized with BCG for 2 weeks）, and BCG ＋Rv3117/DDA/MPL group （immunized with subunit vaccine Rv3117 twice after being immunized with BCG for 2 weeks） （ n =5/group）. The humoral immunity and cellular immunity were evaluated by the enzyme-linked immunosorbent assay （ELISA） and enzyme-linked immunospot Assay （ELISPOT）, respectively. Results The specific antigen Rv3117 of M. tuberculosis was successfully coloned and purified. The results of ELSPOT and ELISA indicated that level of interferon γ （IFN-γ） produced by T lymphocytes of mice of the BCG ＋Rv3117/DDA/MPL group was significantly higher than that of the control group, PBS group, BCG group, and BCG ＋DDA/MPL group （P〈0.05） after being stimulated by the purified protein derivative （PPD） of tuberculin （10.0 lag/mL）. The levels of tumor necrosis factor α （TNF-α）, interleukin 6 （IL-6）, and IL-12p70 of culture supernatants of the BGG ＋DDA/MPL group were significantly higher than those of the control group and PBS group （P〈0.05） and the total level of IgG and IgG1 was also higher. The levels of above indexes of the BCG ＋ Rv3117/DDA/MPL group were significantly better than those of the BCG ＋DDA/MPL group （P〈0.05）. Conclusion The TB subunit vaccine Rv3117/DDA/MPL can strengthen the humoral immunity and cellular immunity of BCG in C57BL/6 mice. The specific antigen Rv3117 of M. tuberculosis is expected to be a novel candidate antigen for designing the TB vaccine and diagnosing the TB.