Development and Evaluation of an Indirect ELISA Based on Recombinant E2 Protein for Detection of Bovine Viral Diarrhea Virus Antibody
[Objective]To provide an effective technique for serological investigation,prevention and control of bovine viral diarrhea(BVD)in China,the E2 protein of Bovine viral diarrhea virus(BVDV)was expressed by eukaryotic system,and used as the coating material to establish a BVDV indirect ELISA antibody detection method for clinic detection.[Method]The BVDV E2 gene was cloned into the eukaryotic expression vector pTT5,and 293F cells were transfected.After 50％of 293F cells died,the cells were collected,ultrasonicated and the recombinant E2(rE2)protein was purified by using Ni ion affinity chromatography.rE2 protein was used as coating antigen,and the conditions of each reaction were optimized,an indirect ELISA method for detection of BVDV antibody was developed,and its specificity,sensitivity,repeatability and conformity with the classical neutralisation test were evaluated.The established method was used for detection of bovine serum samples from different provinces of China.[Result]The rE2 protein was expressed by 293F cells and purified by affinity chromatography,and the purity was 99％with a concentration of 1.74 mg/mL.Through the optimization of each reaction condition,an indirect ELISA antibody detection method for BVDV was successfully established.The method was sensitive enough to detect the weak antibody potency serum(VN=1),which was consistent with that of the commercial kit.It had good specificity and no cross-reaction with the Infections bovine rhinotracheitis virus(IBRV),Bovine parainfluenza virus 3,(BPIV-3),Bovine respiratory syncytial virus(BRSV)and Bovine herpesvirus 1(BHV-1)antibody-positive sera.The coefficients of variation of the intra-and inter-batch reproducibility tests were within 5％,which showed good reproducibility.And the conformity with the classical neutralisation test was 97.5％.Bovine serum samples from different provinces were detected and total 364 sera were positive and 64 sera were negative sera.[Conclusion]The indirect ELISA detection method for antibody against BVDV based on eukaryotic expression of rE2 protein had good specificity and sensitivity,and provided an effective means for the prevention and control of BVD in China.
万方数据
维普
