Establishment Of Indirect ELISA Method Toward N Protein And Epitopes Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome is caused by infection of PRRSV. The disease causes sow fever, anorexia, premature birth, abortion, fetal death and fetal mummies, and the symptom of the all ages especially newborn piglets are reproductive disorders, and respiratory disease. In1987, PRRSV was found in the United States for the first time, and then the virus was isolated in the Netherlands in1991. In1996, the first strain named CH-la was successfully isolated in China. At present, the disease is prevalent in many countries all over the world, causing the huge economic losses to global pig industry. PRRSV is one of the most important virus antigens that threat to the pig industry recently. Now, there are many methods to diagnose PRRSV antibody level, and the mainly method is indirect ELISA in clinical. The N and GP5proteins are coating antigen, the coincidence rate is high in clinical. The coating antigen are purified recombinant N protein and two artificial epitope of N protein (P30and P32) in this research to establish the methods to detection of clinical samples. We also do the research to observe antibody titers of recombinant N protein and two main antigenic epitopes (P30, P32) after artificial infection.The main contents of the study are as following:1. The cloning and expression of ORF7gene of PRRSVThe PRRSV Zhanjiang strain was cultured and total RNA of the virus was extracted. The ORF7gene of PRRSV was amplified and connected into the prokaryotic express plasmid pET32a. The recombinant plasmids were constructed. With expressing by induced and purified, the recombinant N protein were got. The antigenicity of the N protein were analysed by Western blot.2. Establishment of indirect ELISA for antibody detection of PRRSVThe recombinant N protein and artificial epitopes of N protein (P30and P32) were used as antigen for developing indirect ELISA. The optimal coating concentration of the antigen and the dilution of the serum were determined. Through the optimization of the reaction conditions, the indirect ELISA was established. ELISA method of recombinant N protein, P30, P32for detecting antibody of immuned swine serum samples clinical coincidence rate compared with IDEXX commercialization kit were91.5%,88.3%,92.3%; Clinical coincidence rate compared with LSI commercialization kit were88.9%,77.1%,92%; ELISA method for detecting antibody of infected swine serum samples clinical coincidence rate compared with IDEXX commercialization kit were98.3%,95%,98.3%; Clinical coincidence rate compared with LSI commercialization kit were96.7%,90%,98.3%. Clinical coincidence rate of these methods are high, and the P32ELISA is the best among the three methods.