摘要
根据GenBank中的猪肺炎支原体乳酸脱氢酶(LDH)基因序列设计1对引物,PCR扩增LDH基因,首先将扩增片段连接到克隆载体pMD18-T上,然后连接到表达载体pGEX-KG上,经测序正确后诱导表达。重组质粒在大肠杆菌中表达的目的蛋白以可溶性蛋白和包涵体2种形式存在。将超声波破碎的诱导菌液高速离心,其上清用Glutathione Sepharose 4B bead亲和层析纯化。用猪肺炎支原体的标准阳性血清对纯化蛋白作Western-blot检测,出现目的条带;以纯化蛋白为抗原建立了检测猪肺炎支原体抗体的间接ELISA方法,该方法具有较好的稳定性和重复性,较高的特异性与敏感性。用建立的ELISA方法与中国兽医药品监察所研制的IHA试剂盒同时检测120份临床血清,二者总符合率为92.5%。用建立的ELISA方法检测了671份临床送检不同年龄阶段的猪血清,结果显示断奶前仔猪猪肺炎支原体(Mycoplasma hyopnenmoniae,MHP)感染率为44.3%,保育猪为3.0%,育肥猪为17.44%,种猪为73.41%,这初步反映了猪喘气病在各个年龄阶段的感染率。
The truncated LDH gene was amplified from an attenuated Mycoplasma hyopneumoniae strain, ligated to pMD18-T vector for sequence analysis and to pGEX-KG for construction of recombinant expression plsamid which was transformed into E. coli BL21 competent cells. The protein was expressed in the form of solubility and insolubility and was recognized by positive reference serum. The soluble LDH protein was purified using glutathione sepharose 4B bead and used as antigen to develop an indirect ELISA for detection of antibody against Mycoplasrna hyopneumoniae. The cutoff value was determined by testing 69 IHA-negative sera. The comparable result, up to 92.5% agreement, was obtained between our new ELISA and commercial 1HA kit by simultaneously detecting 120 samples. The good reproducibility and specificity were assessed. The assay was further applied to detect 671 sera collected from clinically suspected pigs. The detection rates in sera from postweaning piglets, growing pigs, finishing pigs and adult breeding pigs were 44.3%, 3.0%, 17.44% and 73.41%, respectively, indicating that the prevalence of Mycoplasma hyopneumoniae in pig herds was consistent with previous reports.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2007年第4期511-515,531,共6页
Chinese Journal of Veterinary Science
基金
湖北省科技攻关计划资助项目(2006AA202A05)
