| IBD is a acute contagious disease of little chicks and turkeys which caused by IBDV. It mainly leads to the immunosuppression of poultry, increases the susceptibility to other diseases and decreases the immune response to vaccine. This study aims to construct the recombinant poxvirus which can express the VP2 gene of vvIBDV and analysis its biology characterization, then provide the basis for the development of recombinant vaccines of IBDV. The VP2 protein of wIBDV was expressed with P.Pastoris expression system and its immunogenicity was identified. These provide the basis for the development of sub-unit vaccine and diagnosis reagent. The main contents are as follows:1. Cloning and sequencing analysis for the VP2 gene of HN01 strain of very virulent infectious bursal disease Virus (vvIBDV)Using total RNA of bursa of vvIBDV infectious SPF chicks as templet, the specific gene encoding IBDV VP2 was amplified by RT-PCR. The PCR product was successfully cloned into pMD-18T vector. The construct plasmid pMD-18T-VP2 was screened for the presence of MLUI and NHEI sites by restriction analysis, and the incorporated insert was further verified by dideoxy sequencing. Sequencing Analysis showed that the homology of target gene and animo acid with the standard wIBDV UK661 strain were 98.3% and 99.8% respectively.2. Construction of recombinant fowlpox virus expressing vvIBDV-VP2 and its study of biological characteristicThe gene fragment encoding vvIBDV-VP2 was obtained by PCR. The PCR product was successfully cloned into pEFgpt!2S to construct recombinant fowlpox virus transfer plasmid vector pEFgpt-VP2. The quantity of parental FPV used in transfection and the optimal titer of Mycophenolic Acid (MPA) in selectable culture were determined. The recombinant fowlpox virus transfer plasmid vector pEFgpt-VP2 was cotransfected with fowlpox virus 282E4 strain at CEF by calcium phosphate-DNA coprecipitation method and the target linkedfragment was inserted into ORFl domain of FPV. Then the recombinant virus was selected with selectable culture containing MPA and was purified after 12 passages. Finally, Its specificity was identified positive on its infected CEF through Indirect Immunofluorescence Assay (IFA), this result shows that the recombinant fowlpox virus has successfully expressed the foreign genes and the tandem espressed products have antigenicity; Comparing the TCID50 of the recombinant fowlpox virus (rFPV-IBDV-VP2 ) with that of parental FPV, there was no obvious differences between them. The result shows that insertion of foreign genes into ORFl domain of FPV doesn't affect the propagation and biological characteristics of this virus.The selected recombinant fowlpox virus tandem espressing the wIBDV-VP2 was cultivated for 5 passages in normal culture (2% MEM) and then the liters of the recombinant in the selectable culture (2% MXHAT) and normal culture were tested and compared to the titer of parental FPV in normal culture. Three values were same on the whole, which show that the recombinant was purified and its replication was stable relatively. 3. Secretive expression of wIBDV-VP2 in Pichia yeast and analysis of antigenicityUsing a pair of primers designed according to the relevant nucleotide sequence from GenBank, the specific gene encoding VP2 was amplified by PCR. The PCR product was inserted into the P. Pastoris secretory expression vector pPICZa-A. After the screen of zeocin and the identification of genomic of P. Pastoris, 18 recombinant strains with target gene were obtained. Under the optimal expression conditions, the target protein, which was proved by SDS-PAGE, was expressed in the supernatant of the twelve out of eighteen recombinant strains when induced with methanol. Western-blot and indirect ELISA analysis showed that the expression product had antigenicity.
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