Soluble Expression of PRRSV N Protein and Establishment of Indirect CLIA for Antibody Detection
ZHANG Xian-Wen1,2, ZHOU Long1, TIAN Xin1, CHEN Yu1, LIN Yong-Qiang1, ZHOU Han1, JIKE Wu-Zuo1, LA Qiong3, LI Yan-Min1,*, ZHANG Zhi-Dong1,*
1 College of Husbandry and Veterinary Medicine, Southwest Minzu University, Chengdu 410061, China; 2 Pingchang County Agricultural and Rural Bureau, Pingcang 636499, China; 3 Agriculture and Rural Bureau of Naqu, Naqu 852003, China
Abstract：Porcine reproductive and respiratory syndrome (PRRS), caused by the Porcine reproductive and respiratory syndrome virus (PRRSV), is an acute infectious disease primarily characterized by reproductive disorders in sows (Sus scrofa) and respiratory symptoms in piglets. PRRSV has long been a serious threat to the swine industry in China. The current national control strategy focuses on eradication, making antibody monitoring and prevention of wild-type virus infection crucial for this goal. There is an urgent need to develop a novel, rapid, sensitive, low-cost and fully automated method for detecting PRRSV antibodies. This study established an indirect chemiluminescence immunoassay (CLIA) based on the prokaryotically expressed recombinant nucleocapsid (N) protein of PRRSV. The recombinant expression plasmid pET-28a-N-SUMO was constructed, and the expressed N protein was verified for reactivity via Western blot. The purified recombinant N protein was used as the coating antigen to develop an indirect CLIA for PRRSV antibody detection. The specificity, sensitivity and repeatability of the method were systematically evaluated. Field serum samples were tested in parallel using the established CLIA and a commercial indirect ELISA kit for PRRSV antibody detection to evaluate the concordance between the 2 methods. The results showed that serum samples with OD435 luminous value≥13 438 were considered positive in the CLIA. No cross-reactivity was observed with antisera against other common swine viruses. The repeatability coefficients of variation were all below 7.19%. The detection limit of the CLIA was 4 times higher than that of the commercial ELISA kit. A total of 193 serum samples from Sichuan and Liaoning provinces were tested using both methods, and the Kappa value was 0.924, indicating excellent agreement. These results demonstrated that the established CLIA exhibited high specificity, sensitivity and reproducibility. The entire detection process taked approximately 35 minutes. In conclusion, an indirect CLIA based on the recombinant PRRSV N protein was successfully developed. This method was highly sensitive, reproducible and suitable for rapid detection of PRRSV antibodies in swine serum. This study provides reliable technical support for PRRSV immune monitoring, disease control and eradication efforts.
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