Development Of A Competitive ELISA For Antibody Detection Of North American-type Porcine Reproductive And Respiratory Syndrome And Construction Of Its M Protein Epitope Deletion Strains

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease caused by PRRS virus(PRRSV),which is characterized by reproductive disorders of pregnant sows and respiratory diseases of piglets.PRRS is one of the most serious infectious diseases in China.In view of the lack of effective treatment measures,most pig farms adopt vaccine immunization,increase the level of antibody monitoring,improve the management and feeding methods and other biosafety measures for prevention and control this disease.Therefore,rapid and accurate diagnosis of the disease can be achieved in the early stage of viral infection,and the occurrence and prevalence of PRRS can be controlled to the utmost extent.Based on the current epidemic situation of North American-type PRRSV strains such as highly pathogenic strains,NADC30-like strains in China,and the existing monoclonal antibodies against the M protein of North American PRRSV strains,a competitive ELISA method for rapidly detecting the level of M protein antibody of North American-type PRRSV was researched in this study,and on this basis,a preliminary exploratory study on marker vaccine aganist this monoclonal antibody recogniting epitope was carried out.The main contents of this research include:1.Development and preliminary application of a competitive ELISA for antibody detection of North American-type porcine reproductive and respiratory syndromeA specific competitive-ELISA(C-ELISA)method using purified PRRS whole virus(HuN4-F112 strain)as coating antigen and a monoclonal antibody against the M protein of the North American-type PRRS strain was developed with optimizing the reaction conditions.The results showed that the optimal antigen coating concentration was 660ng/well,the optimal serum dilution was 1:2,and the optimal dilution of the competitive antibody was 1:8.The ELISA result criteria:if the serum sample percentage inhibition(PI)was not less than 25.65%,it was considered to be positive or it was negative.The specificity test showed that this method can only detect the antibody of North American-type PRRS strain and no cross-reaction with those antibody positive serums against PRV,PCV2,CSFV,PEDV,TGEV,European-type PRRS strain.The sensitivity test showed that a 128-fold dilution of positive serum can be detected.And the coefficients of variation(CV)of the intra-and inter-assay repeated tests were less than 10%.Using this method,546 clinical serum samples collected in Heilongjiang region were tested.The positive rate of PRRSV antibody was 95.1%,and the coincidence rate with the commercial IDEXX PRRSV kit was 95.8%.Therefore,the ELISA method established in this study provides an effective detection method for epidemiological investigation of PRRSV,clinical vaccine evaluation and antibody level monitoring before and after immunization.2.Construction of M protein epitope deletion strains of North American-type porcine reproductive respiratory syndrome virusBased on the infectious clone of PRRSV highly pathogenic HuN4-F112 strain and the identified M protein epitope(~3SSLD~6),reverse genetics technique was used to construct the M protein epitope deletion strain with molecular markers.It was proved that three M protein epitope deletion viruses were successfully rescued by cytopathic observation,indirect immunofluorescence assay,Western blotting,deletion and labeling site sequencing and enzyme digestion.The biological characteristics of the rescued viruses and the parent virus,such as virus titer,proliferation kinetics curve,genetic stability,animal experimental antibody production level,were further compared to evaluation of those defective viruses in vitro and in vivo.Although the results show that those M protein epitope deletion viruses can be distinguished from the parent virus in vitro,the results of animal experiments show that the immune serum antibody produced by the constructed M protein epitope deletion virus can not be differentiated by the in vitro ELISA detection method,and the mechanism of antibody production needs to be further explored.This study provides the relevant reference data for the development of effective marker vaccine and lays the relevant research foundation.