牛IgG Fc片段与FnBPB-ClfA串联表达及其对金黄色葡萄菌性奶牛乳腺炎的免疫效果  

Expression of a Fusion Protein Fc-FnBPB-ClfA of Staphylococcus aureus and Its Immune Effect against Cow Mastitis

作　　者：崔健 郝永清 赵红梅 杜琳 李松建 CUI JIan HAO Yong-qing ZHAO Hong-mei DU Lin LI Song-jian(Laboratory of Microbiology and Immunology, College of Veterinary Medicine, lnner Mongolia Agricultural University, Hohhot, lnner Mongolia, 010018, China)

机构地区：[1]内蒙古农业大学兽医学院微生物学与免疫学实验室,内蒙古呼和浩特010018

出　　处：《动物医学进展》2017年第10期27-32,共6页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31460664);农牧交错区生态型现代奶业生产模式研究与示范项目(2012BAD12B09-02)

摘　　要：为了提高金黄色葡萄球菌(S.aureus)黏附素FnBPB-ClfA基因表达蛋白对奶牛的免疫效力,应用RT-PCR扩增奶牛IgG Fc基因片段与构建的金黄色葡萄球菌(S.aureus)FnBPB-ClfA黏附素基因串联构建质粒pMD19T-Fc-FnBPB-ClfA,并通过双酶切将其克隆于pET32a(+),以构建质粒pET32a-Fc-FnBPB-ClfA并进行诱导表达。以盐酸左旋咪唑(LH)或氢氧化铝(ALUM)为佐剂,与纯化的重组蛋白(bFcFnBPB-ClfA)混合以首次免疫肌肉注射,加强免疫乳头管灌注或首次免疫、二次免疫及三次免疫均以肌肉注射方式免疫临近干奶期的奶牛。用间接ELISA和Western blot对各组牛血清中的抗体进行监测,评价免疫效果。结果显示,以LH为佐剂免疫的(A组)牛21d时抗体水平最高,可达到1∶1 600;而以ALUM为佐剂免疫的B组和C组抗体水平分别为1∶800和1∶400。IgG水平动态监测结果表明,以LH为佐剂免疫牛的血清中特异性抗体上升趋势明显高于其他免疫组。研究结果表明,以首次免疫肌肉注射,加强免疫乳头管灌注的方式较好,并且bFc-FnBPB-ClfA与LH佐剂混合后免疫奶牛能最有效激发奶牛免疫反应。The cow IgG Fc gene was fused to FnBPB-ClfA gene by SOE-PCR and cloned into pET-32a （＋） vector to express the recombinant protein of rFc-FnBPB-ClfA in E. coli. Then the lactating cows were immunized with PBS or purified recombinant protein （bFc-FnBPB-ClfA） with the adjuvant levamisole hydrochloride （LH） or aluminum hydroxide （ALUM）. Immunizatioin in the ways of muscle-muscle or muscle- muscle-muscle or muscle-teat canal to the nearly dry period of dairy cows. Indirect ELISA method and Western blot were used to monitor the antibody in the sera of cows in each group. The results showed that the antibody level arrived the highest at 21 d using LH as adjuvant,and could reach 1 ： 1 600,and the anti- body levels of B group and C group were 1 ： 800 and 1 ： 400. The results of this study indicatd that bFc- FnBPB-ClfA and LH adjuvant can effectively stimulate immune response in dairy cows.

关 键 词：金黄色葡萄球菌 奶牛IgG FC 重组蛋白Fc—FnBPB—ClfA 

分 类 号：S857.26[农业科学—临床兽医学] S852.611[农业科学—兽医学]