畜牧兽医学报2018，Vol.49Issue(4)：786-793,8.DOI:10.11843/j.issn.0366-6964.2018.04.016
胞内劳森菌LsaA蛋白的原核表达及间接ELISA抗体检测方法的建立
Prokaryotic Expression of LsaA Protein of Lawsonia Intracellularis and Development of a LsaA-based Indirect ELISA for Antibody Detection
摘要
Abstract
In order to establish a method for detecting specific antibody against Lavsonia intracellularis,the codon-optimized LsaA gene encoding surface protein LsaA of L.intracellularis was cloned into pET32a(+) vector,and the expression of LsaA protein was induced in E.coli host BL21(DE3).After optimization of a series of conditions,an indirect ELISA for detecting antibodies against L.intracellularis was established by using purified LsaA protein as coating antigen.The results showed that the anti-L.intracellularis antibodies were detected specifically by the established ELISA,and there were no cross-reactions with the antibodies against common diarrhea-causing pathogens such as E.coli,Salmonella choleraesuis,porcine epidemic diarrhea virus and transmissible gastroenteritis virus.Serum samples from fecal L.intracellularis-positive pigs which were identified by PCR,were also positive for anti-L.intracellularis antibodies detected by the established ELISA.The ELISA has good sensitivity and repeatability.Anti-L.intracellularis positive serum with 1:800 dilution was still ELISA positive.The intra-and interassay coefficient of variation was 0.352％-2.752％ and 0.877％-3.000％,respectively.Seroepidemiological prevalence of L.intracellularis infection in partial pig farms in Henan province was investigated by using the established ELISA.The results indicated that all the tested pig farms were anti-L.intracellularis antibody positive with positive rate of 13.3％ to 57.1％,with average positive rate of 30.6％.The results showed that the established ELISA in this study can be used for the detection of antibodies against L.intracellularis,and provides a method for the monitoring and epidemiological investigation of L.intracellularis.关键词
猪增生性肠炎/胞内劳森菌/LsaA蛋白/间接ELISAKey words
porcine proliferative enteritis/Lawsonia intracellularis/LsaA protein/indirect ELISA分类
农业科技引用本文复制引用
吴艳阳,杨东东,高冬生,李永涛,常洪涛,王川庆,赵军..胞内劳森菌LsaA蛋白的原核表达及间接ELISA抗体检测方法的建立[J].畜牧兽医学报,2018,49(4):786-793,8.基金项目
国家重点研发计划(2016YFD0501102 （）
2016YFD0500802) （）
河南省高校科技创新团队支持计划(14IRTSTHN015) （14IRTSTHN015）
