猪附红细胞体eno基因重组蛋白的制备及免疫效果研究

为了研究猪附红细胞体eno基因重组蛋白的免疫效果，本试验将猪附红细胞体eno基因与pET-15b载体进行连接，转化大肠杆菌BL21感受态细胞。对表达菌株进行诱导表达、纯化，利用纯化后的猪附红细胞体eno重组蛋白（命名为rMseno）对小鼠进行免疫，从而评价该重组蛋白在小鼠体内产生的免疫应答水平。试验将20只4周龄的雄性昆明小鼠随机分为A、B、C、D 4个组。免疫A组接种纯化后的重组蛋白rMseno；免疫B组接种经IPTG诱导表达后的重组菌E.coli-Mseno；对照C组接种等量PBS；对照D组接种未经过诱导表达的重组菌E.coli-Mseno。通过ELISA检测方法分别测定血清中抗猪附红细胞体特异抗体水平及γ干扰素（IFN-γ）和白细胞介素4（IL-4）细胞因子水平，最后通过脾脏淋巴细胞增殖试验来反映小鼠体内T淋巴细胞的增殖情况。结果显示，构建的重组pET-15b-eno质粒目的基因片段大小为1 632 bp，与预期大小相同；纯化的重组蛋白rMseno大小为61 ku，并能够被鼠抗猪附红细胞体血清所识别；免疫A组和免疫B组小鼠血清中抗猪附红细胞体特异抗体水平，IFN-γ、IL-4细胞因子水平以及T淋巴细胞增殖指数均显著或极显著高于对照组（P<0.05；P<0.01）。结果表明，猪附红细胞体eno基因重组蛋白能够诱导小鼠产生较高的体液免疫水平以及细胞免疫应答水平。

Preparation of Recombinant Protein of Mycoplasma suis eno Gene and Its Immunity Effect

In order to study the immune effect of the recombinant protein of Mycoplasma suis eno gene,the Mycoplasma suis eno gene was linked with pET-15b vector and transformed into E.coli BL21 competent cells.The recombinant Mycoplasma suis eno protein (named rMseno) was used to immunize mice,and the immune response of the recombinant protein in mice was evaluated.Twenty four-week-old male Kunming mice were randomly divided into four groups:Group A,group B,group C and group D.Immune group A was inoculated with recombinant protein rMseno.Immune group B was inoculated with E.coli-Mseno induced by IPTG.Control group C was inoculated with the same amount of PBS.Control group D was inoculated with E.coli-Mseno which was not induced expression.The levels of specific antibody against Mycoplasma suis,IFN-γ and IL-4 cytokines in serum were determined by ELISA,and the proliferation of T lymphocyte in mice was detected by spleen lymphocyte test,the experimental data were analyzed statistically.The results showed that the target gene fragment of the recombinant pET-15b-eno plasmid was 1 632 bp,and the purified recombinant protein rMseno was 61 ku,which could be recognized by the mouse anti-Mycoplasma suis serum.The levels of anti-Mycoplasma suis specific antibody,IFN-γ,IL-4 cytokines and T lymphocyte proliferation index in the sera of mice in immune group A and immune group B were significantly or extremely significantly higher than those in control groups (P<0.05;P<0.01).The results showed that eno gene recombinant protein of Mycoplasma suis could induce high humoral immunity and cellular immune response in mice.