| The monoclonal antibodies(mAbs)against bovine viral diarrhea virus (BVDV)was applied with chicken anti-BVDV egg yolk antibodies(IgY),to develop the method of antigen-captured ELISA(AC-ELISA)for detecting BVDV antigens which were come from clinical feces samples.The optimal concentration of the mAbs and IgY were determined by phalanx titration and the cut-off value,by testing negative feces samples of the healthy cattles.This method was testified by virus isolation and RT-PCR in 23 feces samples.The results showed that the AC-ELISA was highly sensitive and specific in detection of BVDV,this method was 93.75%according with virus isolation and 83.33%according with RT-PCR.By detection system optimization,the detection precision was up to 274ng/mL.It was confirmed that no cross-reaction between bovine Rotavirus and Bovine coronavirus(BCV),but with Classical swine fever virus(CSFV) have cross-reaction weakly.All detection results were in the scope of quality control and accorded with the standardization after control serum test.This kit could be store in 4℃for 6 months;the average coefficient Of variance(CV) of within-run and between-run were 8.37%and 4.31%respectively.The standardization studies of BVDV antigen capture ELISA kit laid foundation for mass-produce.This kit was applied in the investigation for BVDV infection in six cattle farms in north Xinjiang;the result showed that BVDV was widespread in those farms.It appeared that normal herd had been infected BVDV,and this disease had broken out in some cattle farms.The infection rate was up to 100%there.Nine kinds of BVDV isolated from infected cattle farms Xinjiang were cell culture and typed in 5'-UTR,the results showed that the cell culture character accorded with genetic typing,those viruses belonged to non- cell pathological,BVDV-1.Phylogenetic analysis revealed that two isolated viruses belonged to BVDV-1b(Osloss-like group)and one belonged to BVDV-1c(Bega-like group),the others did not belong to any group,it was suspected of a new subgroup.Three E2 gene fragments of BVDV isolated from Shihezi and Manasi in Xinjiang obtained by reverse transcription polymerase chain reaction (RT-PCR).The Sequencing results showed that the fragment length of E2 of shihezi148,manasi and MNS2 were 1121bp,1122bp and 1122bp respectively, they encoded 373,374 and 374 amino acid residue segments,respectively (GenBank accession No.EU159699,EU159703,EU169937 respectively). Sequence comparison and phylogenetic analyses demonstrated that the three isolates fell into the BVDV genotype 1 and were different from other pest viruses such as BVDV genotype2(BVDV-2),Border disease virus(BDV)and Classical swine fever virus(CSFV).The nucleotides sequence of E2 gene of shihezi148 had the highest identity(88.32%,74.42%)with its homologue Australia Bega strain and changchun184 strain,which belonged to the BVDV-1c sub-genotype group,the E2 gene of manasi and MNS had high identity(99.64%,99.22%)with its homologue the live attenuated vaccine strain VEDEVAC,which was an isolated cluster in sub-genotype group BVDV-1b.E2 genes of shihezi148 were cloned into eukaryotic pProEX HTa and pVAX1 procaryotic expression vectors.These two recombinant plasmids were identified by PCR,Enzyme-cutting and sequencing.The result showed that these recombinant plasmids of pproEXhta-E2 and pVAX1-E2 were successfully constructed,pProEX HTa-E2 vector was Transformed to DH5,JM109 and BL21 and induced recombination protein expression,but could not been detected by SDS-PAGE.pVAX1-E2 vector was transected to BHK-21 by GenEscort?, it showed weak green fluorescent protein(GFP) expression, this indirectly proved the transient expression of transfection vector. However, the recombination protein could not detected by SDS-PAGE in cell culture supernatant.The E2 epitopes Manasi strain were predicted by bio-information methods, amino acids (1-118)-( 144-340)-(100-300)- (1-345) epitopes coding region of E2 protein was successfully obtained from recombinant plasmid pMD-E2 by PCR and cloned into prokaryotic expression vector pET-28a, and named pET28a-ED1,pET28a-ED2,pET28a-ED3,pET28a-ED4. These recombinant plasmids were transformed into BL21 competent cells and expressed in high level after being induced with 1 .0mmol / L IPTG, after analyzed by SDS-PAGE. The results showed that the expressed proteins were approximately 18.6KDa,28.2KDa,29.2KDa,43.8KDa in size and accounted for 15% of total bacterial proteins, Western-blot analysis showed that the fusion proteins of 28.2KDa,29.2KDa,43.8KDa were able to react with Bovine polyclonal antibody BVDV serum.pET28a-ED4 recombinant plasmids were transformed into BL21 competent cells, after expressing ,the target protein having been purified and used as antigen in the chess titration test , the optimal circumstance for the ELISA(antigen concentration and sera dilutions) was confirmed. The 43 bovine sera samples was detected used this kit, the result showed that accordance rate is 93.55% with BVDV antibody test kit. Which will provide a good fundament for development of BVDV antibody test kit.
|
PDF Full Text Request