Prokaryotic Expression Of The Major Epitope Regions In NSP2 Of Porcine And Reproductive And Respiratory Syndrome Virus And Development Of An ELISA For Detection Of Antibody

Porcine reproductive and respiratory syndrome(PRRS),characterized by respiratory disease in neonatal pigs and reproductive failure in pregnant sows.The causative agent is porcine reproductive and respiratory syndrome virus(PRRSV),which is a positive-sense RNA virus.NSP2 is the biggest non-structural protein of PRRSV;it is divided into four functional regions,the middle hypervariable region is rich in B cell epitopes,and previous researches had shown that deletion of some B cell epitopes in this region had no effect on PRRSV replication.So deletion the epitopes in NSP2 region will be a good strategy to develop negative marker virus vaccine to support the action for differentiating diagnosis of the virus infected from vaccinated pigs.In this paper,two major B cell epitope regions of NSP2 were expressed in E.coli,and one of the purified recombinant protein was used as the coated antigen to establish an indirect ELISA method for the detection of PRRSV antibody.Based on the cloned NSP2 gene of GSWW strain,the NSP2 fragment of 2010-2759 bp(named EF2)was amplified using a pair of specific primers,and then subcloned into pET-28 a vector for expression.The fragment corresponding to NSP2 2226-3110 bp(named GD2)was synthesized and subcloned into pET-30 a for protein expression.Two recombinant vector were transformed into Escherichia coli for protein expression.The molecular weight of expressed EF2 and GD2 are 29 and 47 ku,respectively.Western blotting showed that the recombinant proteins have high reaction activity and could be recognized by positive serum of PRRSV.EF2 protein was used to coating ELISA plate to develop an indirect ELISA for detection antibodies against NSP2 of PRRSV.The key parameters of this ELISA are as following: optimal coating concentration of EF2 is 1 ??/ mL,the optimal of dilution of the serum and HRP labeled Rabbit anti-Pig IgG is 1?20 and 1?2000,respectively.By detection of 30 serum samples from PRRSV uninfected pigs,the cut-off value was determined by calculating the ratio of OD value of sample to positive control(S/P).If the S/P value is more than 0.17,it is considered to be positive for NSP2 antibody;if the S/P value is less than or equal to 0.17,it is considered to be negative for NSP2 antibody.According to above criteria,this EF2-ELISA test show a good specificity for detecting 30 samples from PRRSV uninfected pigs and no cross reactivity with CSFV,PRV,FMDV positive serum.By detectcting 133 sera from PRRSV infected pigs,the antibody positive ratio is 88.7%,indicating a good sensitivity and specificity of the EF2-ELISA.The coefficients of variation for intra-assay and inter-assay is 3.8%~6.8% and 4%~8.6% respectively.Total of 365 serum samples from infected pigs were detected by this ELISA and compared with two commercial ELISA kits,the total coincidence rate is 73.1%;(LSI)and 77.2%(Yuanheng),respectively.Nine monoclonal antibodies(MAbs)were prepared using the GD2 protein as antigen.These 9 MAbs were detected to recognize 4 epitopes in GD2 protein by superposition ELISA.Four MAbs were prepared in mouse that recognize 4 different epitopes in NSP2.Western blotting showed these 4 MAbs can recognize both EF2 and GD2 proteins.The preparation of MAbs make a good foundation for establishing ELISA based on these MAbs in the future research.