中国兽医科学2012，Vol.42Issue(5)：495-500,6.
猪札幌病毒VP1基因的表达及ELISA抗体检测方法的建立
Expression of VP1 gene of porcine sapovirus and development of an indirect ELISA for the detection of antibodies against porcine sapovirus
摘要
Abstract
The aim of the present study was to establish an indirect ELISA method for the detection of porcine sapovirus antibody based on the expression of VP1 gene of porcine sapovirus in Escherichia coli.Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank,the VP1 gene of porcine sapovirus was amplified with RT-PCR method from stool samples of piglet suffered from diarrhea.The PCR product was then cloned into a pET-28a（＋） vector to construct a prokaryotic recombinant plasmid pETSAVCAP.The pETSAVCAP was transformed into E.coli BL21（DE3） for expression.Then an indirect ELISA was established to detect antibody against porcine sapovirus with the VP1 protein,which was purified by SDS-PAGE,as coating antigen.The VP1 protein was stably and highly expressed and manifested good reactinogenicity as was confirmed by Western-blot.The optimum working concentration of antigen was 0.5 μg in 100 μL per well.By using this novel ELISA approach,490 pig serum samples which were collected from six provinces of China were tested with a positive rate of 65.31% of porcine sapovirus antibodies.The method was specific,sensitive and reliable.The results indicated that the ELISA method based on the purified recombinant protein could be used to detect antibodies against porcine sapovirus.关键词
猪札幌病毒/VP1基因/表达/酶联免疫吸附试验/抗体/检测Key words
porcine sapovirus/VP1 gene/expression/enzyme-linked immunosorbent assay/antibody/detection分类
农业科技引用本文复制引用
李润成,刘国华,黄泽彬,丁建,汪镇南,肖朝庭,余兴龙..猪札幌病毒VP1基因的表达及ELISA抗体检测方法的建立[J].中国兽医科学,2012,42(5):495-500,6.基金项目
湖南省科技重大专项 （）
湖南农业大学人才引进基金项目 （）
