猪肺炎支原体P46-P65重组蛋白的表达及间接ELISA抗体检测方法的建立  

Expression of Mycoplasma hyopneumoniae P46-P65 recombinant protein and establishment of an indirect ELISA for antibody detection

作　　者：杨振宇 李璇[1,2] 刘一宁 谢邵波 郑金 刘春燕[1,2] 林美婷 刘腾 唐红剑 余兴龙 YANG Zhen-yu;LI Xuan;LIU Yi-ning;XIE Shao-bo;ZHENG Jin;LIU Chun-yan;LIN Mei-ting;LIU Teng;TANG Hong-jian;YU Xing-long(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Combetter Biotechnology Co.,Ltd.,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]湖南康保特生物科技有限公司,湖南长沙410128

出　　处：《中国预防兽医学报》2024年第7期709-715,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：湖南省重点领域研发计划项目(2022NK2049)。

摘　　要：为建立猪肺炎支原体(Mhp)血清学调查及免疫评估方法,本研究利用DNAStar生物学软件对Mhp的P46和P65进行抗原表位分析,利用重叠延伸PCR(SOE-PCR)获得P46-P65融合基因,构建重组表达质粒pETP46-P65,将其转化大肠杆菌BL21(DE3)感受态细胞,经诱导后获得了可溶性表达的重组P46(aa33~aa419)-P65(aa307~aa627)蛋白(rP46-P65)。以纯化的r P46-P65为包被抗原,经优化各反应条件后建立了检测Mhp抗体的间接ELISA方法。特异性试验结果显示所建立的方法与CSFV、FMDV、PEDV、PCV2、PRV、PRRSV和APP等阳性血清均无交叉反应。该方法可检测到最高稀释至6 400倍的Mhp阳性血清,批内和批间变异系数均小于5%。利用IDEXX试剂盒和本研究建立的方法同时检测298份临床血清样品,前者的检测阳性率为62.4%(186/298),后者的检测阳性率为73.8%(220/298),两者检测结果的总符合率为88.6%。IDEXX检测为阳性的血清,采用本研究建立的ELISA方法检测也均为阳性;而部分Mhp阳性猪经IDEXX试剂盒检测为阴性的血清,该ELISA方法检测结果却为阳性,其中79.4%(27/34)检测结果有差异的血清经Mhp颜色变化试验证明均为阳性,表明本研究建立的ELISA方法的敏感性明显高于IDEXX方法。本研究建立的检测猪Mhp抗体的间接ELISA方法与目前广泛使用的方法相比优势明显,为临床血清流行病学调查及血清抗体水平评估提供了可行方法。To establish a method for serological investigation and evaluation of vaccination against Mycoplasma hyopneumoniae(Mhp),the present study utilized DNAStar biological software to analyze the epitopes of Mhp-P46 and Mhp-P65.The P46-P65 fusion gene was obtained by overlapping extended PCR (SOE-PCR),and the expression plasmid pETP46-P65 was constructed and transformed into Escherichia coli BL21 (DE3) receptor cells.Soluble expression of recombinant P46(aa33-aa419)-P65(aa307-aa627)(r P46-P65) protein was obtained after induction.Using the purified r P46-P65 protein as coated antigen,an indirect ELISA method was established for the detection of Mhp antibody after optimizing the reaction conditions.The specific experimental results showed that the proposed method demonstrated high specificity,showing no cross-reaction with sera positive for other pathogens,including CSFV,FMDV,PEDV,PCV2,PRV,PRRSV,APP.The assay was able to detect Mhp antibodies when serum samples were diluted up to 6400times,and the coefficient of variation within and between batches was less than 5%.Moreover,two hundred and ninety-eight clinical serum samples from farm herds were detected simultaneously by IDEXX kit and the i ELISA.The positive rate of IDEXX kit was 62.4%(186/298) and the positive rate of i ELISA was 73.8%(220/298),and the concordance rate of the two methods was 88.6%.All IDEXX-positive samples were also positive in the newly established i ELISA and IDEXX-negative samples were tested negative with the i ELISA.However,some positive serums from the same herds in the assay of the i ELISA were negative by means of IDEXX assay,and79.4%(27/34) of the serums with different results were also confirmed positive by the Mhp metabolic inhibition test.The results indicates that the sensitivity of the i ELISA is significantly higher than that of IDEXX method.The above results show that the i ELISA established in this study has significant advantages over currently available methods,and would be useful in the future for clinical investigations of serum

关 键 词：猪肺炎支原体 重组 融合蛋白P46-P65 间接ELISA 

分 类 号：S852.62[农业科学—基础兽医学]