目的  在原核系统中表达金黄色葡萄球菌（金葡菌）锰转运蛋白C（manganese transporter C，MntC），对其免疫原性进行分析，为筛选金葡菌疫苗候选抗原提供参考。方法  合成MntC基因，克隆至pET-22b（+）载体，构建重组表达质粒pET-22b-mntc，转化至E. coli BL21（DE3），异丙基硫代–β–D–半乳糖苷诱导表达，镍柱纯化。将纯化产物进行免疫印迹法鉴定，再采用分子排阻高效液相色谱法进行纯度分析。将铝佐剂吸附重组MntC，免疫BALB/c小鼠作为实验组，将铝佐剂免疫BALB/c小鼠作为对照组。经ELISA检测小鼠血清中特异性IgG抗体水平、中性粒细胞呼吸爆发试验检测小鼠血清特异性抗体功能，再用细胞因子试剂盒检测小鼠脾淋巴细胞培养上清液中抗原特异性细胞因子水平。免疫后小鼠用致死剂量金葡菌49521菌攻毒，观察小鼠存活情况。结果  经双酶切及测序鉴定，重组表达质粒pET-22b-mntc构建正确。重组MntC相对分子质量约为34 000，以可溶性形式表达，可与抗His单克隆抗体特异性结合，纯度大于95%。将铝佐剂吸附MntC免疫小鼠可诱导产生高滴度特异性IgG抗体；经MntC免疫的小鼠血清能明显增强中性粒细胞对49521菌株的呼吸爆发，实验组在60 min内发光值均值比对照组提高78%；实验组脾淋巴细胞培养上清液中抗原特异性IFN-γ、IL-5和IL-17A浓度分别为对照组的5.8、9.9和62.8倍，均高于对照组且差异有统计学意义（t值分别为3.75、3.95和3.93，P值分别为0.004、0.003和0.003）；MntC免疫小鼠后以致死剂量49521菌株攻毒，小鼠生存率从1/6提升至2/3。结论  正确表达的重组MntC具有较好的免疫原性，可作为金葡菌疫苗的候选抗原组分。

Objective  To express the manganese transporter C (MntC) of Staphylococcus aureus in prokaryotic system, analyze its immunogenicity, and provide reference for screening candidate antigens of S. aureus vaccine. Methods  The MntC gene was synthesized and cloned into pET-22b(+) vector. The constructed recombinant plasmid pET-22b-mntc was transformed into E. coli BL21 (DE3) and induced by isopropylthio-β-D-galactoside. The expression product was purified by nickel column, and analyzed by immunoblot and size exclusion high performance liquid chromatography. BALB/c mice immunized with aluminum adjuvant-adsorbed recombinant MntC were used as the experimental group and BALB/c mice immunized with aluminum adjuvant were used as the control group.The level of specific IgG antibody in BALB/c mice sera was detected by ELISA, the function of these specific antibodies was detected by neutrophil respiratory burst test, and the levels of antigen-specific cytokines in BALB/c mice spleen lymphocyte culture supernatant were detected by cytokine kits. After immunization, the mice were challenged with lethal dose of S. aureus strain 49521,and observed for survival. Results  Restriction enzymes digestion analysis and sequencing confirmed that recombinant expression plasmid pET-22b-mntc was constructed correctly. Recombinant MntC, with relative molecular mass of about 34 000, was expressed in a soluble form. Purified MntC specifically bound to anti-His monoclonal antibody, and the purity was more than 95%. High titer IgG antibodies were induced in immunized mice by aluminum adjuvant adsorbed recombinant MntC. Compared with those in control group, the serum of MntC immunized mice significantly enhanced the respiratory burst of neutrophils against strain 49521, and the mean luminescence value of the experimental group was 78% higher than that of the control group within 60 min. The levels of antigen-induced IFN-γ, IL-5 and IL-17A in the mice spleen lymphocyte culture supernatant of the experimental group were 5.8, 9.9 and 62.8 times of those in the control group, respectively, which were statistically significantly higher (t values were 3.75, 3.95, 3.93, and P values were 0.004, 0.003, 0.003, respectively). The survival rate of MntC immunized mice increased from 1/6 to 2/3 after being challenged with lethal dose of strain 49521. Conclusion  The correctly expressed recombinant MntC shows good immunogenicity and may be used as a candidate antigen component for S. aureus vaccine.