The purpose of this study was to develop a method for detecting antibodies against porcine pseudorabies virus （PRV）. According to the published genomic sequence of PRV SA strain, an approximately 1 070-bp gD gene fr...The purpose of this study was to develop a method for detecting antibodies against porcine pseudorabies virus （PRV）. According to the published genomic sequence of PRV SA strain, an approximately 1 070-bp gD gene fragment was amplified by PCR. The PCR products were cloned into the prokaryotic expression vector pET30a and the positive recombinant plasmid was transformed into E. coli BL21. Through induction with IPTG, the recombinant gD protein was expressed as inclusion bodies. As analyzed by western blot assay, the purified recombinant gD protein had good antigenicity and high specificity. Using the purified gD protein as coating antigen and horseradish peroxidase labeled staphylococcal protein A （PPA） as secondary antibody, we developed a PPA-ELISA for detecting antibodies against porcine PPV. No cross-reaction with the positive sera against seven common pathogens in pigs including classical swine fever virus, porcine parvovirus, porcine reproductive and respiratory syndrome, Japanese encephalitis virus, porcine circovirus type 2, porcine epidemic diarrhea virus, transmissible gastroenteritis virus was observed. The repeatability test showed that the intra- and inter-assay coefficients of variation were lower than 5% and 10%, respectively. Compared with the ELISA gD antibody test kit produced by IDEXX, the coincidence, sensitivity and specificity of the developed PPA-ELISA were 92.0%, 95.1% and 88.1%, respectively. The developed PPA-ELISA had good repeatability, sensitivity and specificity and was a rapid and simple serological method for surveillance of PRV antibodies in pig herds as well as for rapid diagnosis and epidemiological investigation of PRV infection.展开更多
基金funded by the Special Fund for Research and Development of Application Technology of Binzhou City(200706)Youth Science and Technology Innovation Fund of Shandong Binzhou Animal Science & Veterinary Medicine Academy (2007-02)
文摘The purpose of this study was to develop a method for detecting antibodies against porcine pseudorabies virus （PRV）. According to the published genomic sequence of PRV SA strain, an approximately 1 070-bp gD gene fragment was amplified by PCR. The PCR products were cloned into the prokaryotic expression vector pET30a and the positive recombinant plasmid was transformed into E. coli BL21. Through induction with IPTG, the recombinant gD protein was expressed as inclusion bodies. As analyzed by western blot assay, the purified recombinant gD protein had good antigenicity and high specificity. Using the purified gD protein as coating antigen and horseradish peroxidase labeled staphylococcal protein A （PPA） as secondary antibody, we developed a PPA-ELISA for detecting antibodies against porcine PPV. No cross-reaction with the positive sera against seven common pathogens in pigs including classical swine fever virus, porcine parvovirus, porcine reproductive and respiratory syndrome, Japanese encephalitis virus, porcine circovirus type 2, porcine epidemic diarrhea virus, transmissible gastroenteritis virus was observed. The repeatability test showed that the intra- and inter-assay coefficients of variation were lower than 5% and 10%, respectively. Compared with the ELISA gD antibody test kit produced by IDEXX, the coincidence, sensitivity and specificity of the developed PPA-ELISA were 92.0%, 95.1% and 88.1%, respectively. The developed PPA-ELISA had good repeatability, sensitivity and specificity and was a rapid and simple serological method for surveillance of PRV antibodies in pig herds as well as for rapid diagnosis and epidemiological investigation of PRV infection.
