| 猪肺炎支原体乳酸脱氢酶基因的克隆、表达、纯化及其间接ELISA检测方法的建立 |
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| 引用本文： | 姚睿玉,何启盖,周锐,陈焕春.猪肺炎支原体乳酸脱氢酶基因的克隆、表达、纯化及其间接ELISA检测方法的建立[J].中国兽医学报,2007,27(4):511-515,531. |
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| 作者姓名： | 姚睿玉 何启盖 周锐 陈焕春 |
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| 作者单位： | 华中农业大学动物医学院湖北省预防兽医学重点实验室,农业微生物学国家重点实验室,湖北,武汉,430070 |
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| 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 Sepharose4Bbead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试荆盒同时检测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。
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| 关 键 词： | 猪肺炎支原体 乳酸脱氢酶基因 抗体检测 诊断 |
| 文章编号： | 1005-4545（2007）04-0511-05 |
| 修稿时间： | 2005-09-28 |
Cloning,expression of Mycoplasma hyopneumoniae LDH gene and development of indirect ELISA for antibody detection |
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| YAO Rui-yu,HE Qi-gai,ZHOU Rui,CHEN Huan-chun.Cloning,expression of Mycoplasma hyopneumoniae LDH gene and development of indirect ELISA for antibody detection[J].Chinese Journal of Veterinary Science,2007,27(4):511-515,531. |
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| Authors: | YAO Rui-yu HE Qi-gai ZHOU Rui CHEN Huan-chun |
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| Institution: | College of Veterinary Medicine, Huazhong Agricultural University, The Provincial Key Laboratory of Preventive Veterianry Medicine, The National Key Laboratory of Agricultural Microbiology, Wuhan 430070, China |
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| Abstract: | 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. |
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| Keywords: | ELISA |
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