绵羊肺炎支原体P113蛋白C末端基因真核表达载体的构建及其小鼠免疫应答  

Construction of eukaryotic expression vector for C terminal gene of Mycoplasma ovipneumoniae P113 protein and its immune response in mice

作　　者：杨鹏 吴燕[1] 岳筠[2] 陈静 李梅 王慧[2] 张双翔[2] 文明[1,3] 程振涛 YANG Peng;WU Yan;YUE Jun;CHEN Jing;LI Mei;WANG Hui;ZHANG Shuangxiang;WEN Ming;CHENG Zhentao(College of Animal Science,Guizhou University,Guiyang 550025,China;Guizhou Center for Animal Disease Prevention and Control,Guiyang 550008,China;Guizhou Key Laboratory of Animal Disease and Veterinary Public Health,Guiyang550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病预防控制中心,贵州贵阳550008 [3]贵州省动物疫病与兽医公共卫生重点实验室,贵州贵阳550025

出　　处：《中国兽医学报》2022年第3期496-501,521,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31660723,32060786);贵州省科技计划资助项目(黔科合基础[2019]1181号);黔科合支撑计划资助项目([2021]一般161号);贵州省动物疫病预防控制中心人才基地建设资助项目(黔疫控[2019]06号)。

摘　　要：为探究绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)P113蛋白小鼠免疫应答情况。构建了Mo P113蛋白C末端基因pVAX1-P113表达质粒,转染至MDBK细胞,应用PCR技术及间接免疫荧光试验评价其体外表达效果;免疫实验动物BALB/c小鼠,通过间接ELISA方法分析重组质粒诱导小鼠特异性抗体和细胞因子(IL-2、IL-4和IFN-γ)分泌情况。结果显示,成功构建含Mo P113基因重组真核表达质粒pVAX1-P113,重组质粒转染MDBK细胞能有效转录并表达目的蛋白;小鼠免疫试验结果显示重组质粒pVAX1-P113免疫小鼠后血清特异性抗体水平显著升高,在免疫7周时血清特异性抗体仍为阳性;血清中IL-2、IL-4和IFN-γ的分泌水平显著升高,并在时间上呈先上升后下降的趋势;在不同试验组之间特异性抗体、IL-2、IL-4和IFN-γ的分泌量也存在显著性差异,pVAX1-P113(100μg)组分泌水平显著高于其他试验组。结果表明,重组质粒pVAX1-P113能诱导免疫小鼠产生免疫应答;为羊支原体肺炎(MPSG)核酸疫苗研究与应用提供了依据。To explore the immune response of Mycoplasma ovipneumoniae(Mo) P113 protein in mice, the expression plasmid pVAX1-P113 of the C-terminal gene of the P113 protein of Mycoplasma ovipneumoniae was constructed and transfected into MDBK cells, and its expression in vitro was evaluated by PCR and indirect immunofluorescence assay.BALB/c mice were immunized and the secretion of mouse-specific antibodies and cytokines(IL-2,IL-4 and IFN-γ) induced by recombinant plasmid was analyzed by indirect ELISA method.The results showed that the recombinant eukaryotic expression plasmid pVAX1-P113 containing the C-terminal gene of the Mo P113 was successfully constructed.The recombinant plasmid transfected into MDBK cells can effectively transcribe and express the target protein.the results of mouse immunization test showed that the level of serum specific antibody increased significantly after immunization with recombinant plasmid pVAX1-p113,and the serum specific antibody was still positive after 7 weeks of immunization;the secretion levels of IL-2,IL-4 and IFN-γin the serum increased significantly,and showed a trend of first rising and then falling in time.There were also significant differences in the secretion of specific antibodies,IL-2,IL-4 and IFN-γbetween the different test groups.The secretion level of the pVAX1-P113(100μg)group was significantly higher than that of the other test groups.The results show that the recombinant plasmid pVAX1-P113 can induce immune responses in immunized mice.This study provides basic research data for the research and application of Mycoplasma pneumonia of sheep and goats(MPSG)nucleic DNA vaccine.

关 键 词：绵羊肺炎支原体 P113蛋白 DNA疫苗 免疫应答 

分 类 号：S852.62[农业科学—基础兽医学]