| Porcine bocavirus(PBo V)was found in Sweden in 2009 for the first time. So far it is not clear for its transmission mode,infection mechanism and the function of genome. While in investigation of epidemiology,some researchers found that PBo V had higher positive rate in swine which infected postweaning multisystemic wasting syndrome(PMWS). In addition,another scholars proposed that PBo V may cause infection with pathogen of respiratory disease. Now,most people think its genome encodes three open reading frames(ORFS) corresponding to the non-structural protein NS1,antigen protein VP,and nonstructural proteins NP1,VP protein is the major antigenic protein of PBo V,and shows a very high degree of variability. Non-structural proteins NP1 encodes a relatively conservative and highly phosphorylated non-structural proteins. Recently,there is no improved approched to test PBo V like serological methods in the world except some defective PCR detection methods,thus effectively serological methods are extremely required. This study extract PBo V from sick pigs in Hebei, prediction analysis is used in B cell liner epitope about NP1 and VP2 gene,clone and express conservative NP1 gene and relatively truncated conservative VP2 gene,then establish a rapid detection of PBo V serological diagnostic methods according to optimizing and compare as purified protein NP1 and VP2 as coating antigen successfully.According to the gene sequences of NP1 and VP2 published in Gen Bank,a pair of specific primer was designed. NP1 and truncated VP2 gene sequences were amplified by polymerase chain reaction.Then,vector and target fragment with restriction enzyme cutting site were ligated and transferred. Named the recombinant plasmids with PMD-NP1 and PMD-VP2 respectively. After double enzyme digestion of PMD-NP1 and PMD-VP2, connected with prokaryotic expression vector p ET32 a. p ET32a-NP1 and p ET32a-VP2 expression plasmids was constructed. Induced by IPTG and run on a SDS–PAGE gel, 44 ku and 35 ku protein were detected. They were consistent with expected protein molecular weight and existed in the supernatant in solubility form. At the same time the protein enriched in inclusions form. Making use of the his label in prokaryotic expression vector, p ET32a-NP1 and p ET32a-VP2 recombinant proteins were purified. Western-blotting result showed that the recombinant protein and PBo V positive serum had good reactogenicity. The molecular weight of target protein were 44 ku and 35 ku.Establwashing indirect ELISA for detection of PBo V antibodies and comparing two ELISA methods for detection according to optimized their reaction condition as two purified recombinational proteins as coating antigen. Optimized result showed that as protein NP1 as coasting antigen, the most concentration was 4ug/m L; confining liquid was 2% gelatin; the most dilutability of positive and negative serum were 1:80, the most dilutability of second antibody was 1:1000,substrate developing time was 10 min. This method did not cause cross reaction with CSFV, PRRSV, PPV and PCV2. Coefficient of variation were all less than 10% in repeatability of intra-assay and inter-assay, howeve,as protein VP2 as the coasting antigen,the most concentration was 2ug/m L. Blocking Buffer is 2% gelatin. The optimal dilutions of positive and negative serum were 1:80.The optimal dilution of HRP-Ig G was 1:1000.Chromogenic substrate time was 10 min. There was no evidence of cross-reactivity with CSFV, PRRSV and PPV positive seras. The inter-assay and the intra-assay CVs were less than 10% either. It indicated that this method had higher sensitivity and repeatability. By comparison,the ELISA coating with NP1 protein had higher detection ratio than coating with VP2 protein. |
