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Establishment Of ELISA Method For Detection Of Porcine Reproductive And Respiratory Syndrome Virus Antibodies

Posted on:2020-09-03Degree:MasterType:Thesis
Country:ChinaCandidate:H S ChenFull Text:PDF
GTID:2370330575961236Subject:The vet
Abstract/Summary:PDF Full Text Request
Porcine Reproductive and Respiratory Syndrome(PRRS)is a strong contact viral infection that causes reproductive and respiratory disorders in pigs and directly causes significant economic losses in the breeding industry.Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)is a highly toxic strain with rapid variability and immunosuppressive and persistent infection characteristics that make it difficult to control.The prevention and control of PRRS is mainly controlled by the vaccination of vaccines and the safety of biosafety.The application of the PRRSV antibody detection kit can well detect PRRSV antibodies in pig serum,monitor the prevalence of porcine reproductive and respiratory syndrome,and evaluate the immune effects of related vaccines.In order to develop a PRRSV antibody detection method with better specific sensitivity,to better realize the epidemiological monitoring and diagnosis of PRRSV and evaluate the immune effect of the relevant vaccine,this study cloned the PRRSV GDr180 strain N protein gene sequence into pET-32 a In the vector,the recombinant cloning vector pET-32A-N was successfully constructed and transformed into E.coli BL21(DE3).By exploring the conditions of induced expression,the N protein was highly expressed in soluble expression mode,and the expression product was positively reacted and positively correlated with PRRSV GDr180 positive serum by Western blot.By exploring the protein purification conditions,it was finally determined that the eluate containing 100 mM imidazole could elute the higher purity N protein,and the N protein concentration of the kit was determined to be 250 ?g/mL using the BCA protein concentration.The purified N protein was used as the coating antigen of the PRRSV antibody indirect ELISA assay,and the parameters and reagents were optimized.52 established PRRSV antibody-negative pig serum was detected by the established ELISA method.The average value(x)and standard deviation(s)of the S/P value were calculated according to the OD450 value of the negative serum sample,Combined with the statistical principle,it was determined that the critical value S/P value was ?0.172,and the S/P value was <0.085,which was negative,which was suspicious between the two.The specificity,repeatability and sensitivity of the method were tested.The results showed that the established PRRSV antibody detection method was used to detect Pseudo Rabies Virus(PRV),Classical Swine Fever Virus(CSFV),Mycoplasma Hyopneumoniae(MHP),Porcine parvovirus(PPV),Japanese Encephalitis Virus(JEV),porcine circovirus 2(PCV2),Haemophilus parasuis(HPS)and other positive sera,the SP value is less than 0.085,which proves that the established ELISA method is specific.Good in the in-batch and inter-assay repeated tests,the coefficient of variation between each test serum test value is less than 10%,further indicating that the ELISA method has good repeatability;in the sensitivity test,the test shows that The results of 80-fold dilution of strong and weak positive serum were still positive,and the test showed that the established ELISA method was sensitive.Finally,196 serum samples were simultaneously detected by the indirect ELISA method and the IDEXX PRRSV ELISA antibody detection kit established in this experiment.Compared with the results of the IDEXX ELISA test kit,the indirect ELISA method established in this experiment is relatively specific to 96%.The agreement between the two reached 91%.In summary,the indirect ELISA antibody assay established in the study can be used to detect PRRSV antibodies in pig serum,thereby monitoring the prevalence of porcine reproductive and respiratory syndrome and evaluating the immune effects of related vaccines.
Keywords/Search Tags:Porcine reproductive and respiratory syndrome, Prokaryotic expression, ELISA, N protein
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