Establishment And Preliminary Application Of An Indirect ELISA For The Detection Of Antibody Against Bovine Parainfluenza Virus Type 3

Bovine parainfluenza virus type 3(BPIV3)is an enveloped, non-segmented, negative-sense,single-stranded RNA virus, and can cause bovine respiratory syndrome( BRDC) with other pathogen. It mainly cause upper respiratory infections of adult cattle and calves and the main clinical features is bronchitis, pneumonia and other respiratory symptoms, the serious animal even can cause miscarriage. It has brought huge losses in cattle industry.At present, the research of BPIV3 is still poor in China. There is a not particularly effective commercialization detection kit, and considering the powerful communicating ability and great harm of BPIV3, it is imminent to establish a convenient, accurate detection method of BPIV3. The study found that NP protein of BPIV3 is one of the highest concentration proteins in BPIV3 and it is highly conserved, there are three major epitopes on it, two located of them in the C-terminus.HN protein is a surface antigen and highly conserved during evolution. The HN and NP proteins have a good ability to detect antigen. Using the epitope genes of HN and NP protein in series, as a detection antigen, can amplify detection in better principle.In this study, we analyzed amino acid sequencing of the NP and HN protein of SD2014BPIV3 by Lasergene software and combining literature. The dominant antigenic region, that C-terminal 388aa~515aa of NP protein and C-terminal 355aa~572aa of HN protein, was determined. Selecte NP and HN protein epitope and make it in series(named NP-HN). p ET28a(+)-NP-HN vector of prokaryotic expression was constructed. We optimized the ELISA reaction conditions using the NP-HN recombinant protein as coating antigen, and established an i ELISA method to specifically detect the positive serum of BPIV3. Further the i ELISA method was compared with virus neutralization test and imported ELISA kits, respectively.The specific content and results are as follows:1.This study successfully cloned truncated NP-HN gene and created p ET28a( +)-NP-HN prokaryotic expression plasmid, and the plasmid was successfully transformed into E.coli BL21(DE3), IPTG induced the BL21 the result shows that the target protein(NP-HN fusion protein)was abundantly expressed, and the size is 44 k Da.2.Culture large numbers of recombinant bacteria and induce to get abundance target protein,application His-tag nickel column affinity chromatography purified protein. Higher verify purity ofthe purified protein by SDS-PAGE. We use western blot to analysis the NP-HN fusion protein, the result shows that the purified protein can recognize BPIV3 positive serum. The purified NP-HN protein is detected by Pierce BCA protein quantitation Kit, the protein concentration is 900 μg/ml.3.Using purified NP-HN protein immunize New Zealand white rabbits for three times to get polyclonal antibodies that serum titer is 1:32000. Using this polyclonal antibody to test BPIV3,BVDV and IBRV full virus by ELISA respectively and the results showed that polyclonal antibodies can react with BPIV3, but not with BVDV and IBRV, the result suggested the purified NP-HN protein has good immune response and specificity. The polyclonal antibody was detected by western blot results showed that the NP and HN protein of full virus is capable to bind with polyclonal antibodies. It also indirectly described that fusion protein NP-HN epitopes has not changed during expression and purification process, and has biological activity that can be used as a detecting antigen of indirect ELISA method.4.Establishment of an indirect ELISA for the detection of antibody against BPIV3Taking the purified recombinant NP-HN protein as antigen, we established indirect ELISA method for detecting BPIV3 antibody. To validate the assays, the ELISA was performed with standard positive and negative sera of BPIV3. Modified reaction condition of ELISA: Antigen concentration was 4ug/ml and should be coated overnight at 4℃; Blocking solution was 20% horse serum, and should closure for 1.5h; Serum dilution of 1:50, incubation time for 1h; Rabbit anti-bovine-HRP dilution of 1:4000, the incubation time for 1h; TMB coloration for 10 min, 1M H2SO4 terminated the reaction, then use micro plate reader to determined OD450 nm. The criterion of this study is that the titer less than 0.3 are negative, greater than 0.3 is positive. The NP-HN protein had no cross reaction with positive serum of bovine viral diarrhea virus and infectious bovine rhinotracheitis virus. The coefficients of variation for intra and inter-assay were lower than8%. Further the i ELISA method was compared with virus neutralization test and imported ELISA kits, respectively. The total coincidence rate with virus neutralization test and imported ELISA kit were 96.67% and 95.56%.5. The total positive rate of 270 clinical serum samples from Shandong, Liaoning, and Tianjin was 82.59%.Indicating that there is infection and epidemic of BPIV3 above cattle field.