Construction and expression of recombinant adenovirus vector against infectious pancreatic necrosis with VP2 protein and VP3 protein of Atlantic salmon
Infectious pancreatic necrosis(IPN)was an infectious disease caused by infectious pancreatic necrosis virus(IPNV),which mainly affected salmonids.To clone the infectious pancreatic necrosis virus(IPNV)VP2 gene and VP3 gene,constructed their recombinant adenovirus vector.In this study,the IPNV VP2 gene and VP3 gene were amplified and inserted into the pAd-Track-CMV vector by PCR and multi-gene fragment homologous re-combination technology.After linearization,the recombinant plasmids were recombined with the pAd-easy-1 vec-tor in E.coli BJ5183 to construct a recombinant adenovirus plasmid,which was identified by PCR and digestion with NotⅠand HindⅢ,and then transfected into 293T cells to obtain the recombinant adenovirus after linearizing with PacⅠ.The viral replication was monitored using the expression of green fluorescent protein(GFP).The ex-pression of VP2 protein and VP3 protein were confirmed by the Western-blotting method,and the recombinant viral titer was detected.The results indicated that the IPNV VP2 gene and VP3 gene were cloned with a length of 2 211 bp,and the recombinant adenovirus vectors for the target genes were constructed.The virus could express a molecular mass of about 54 kD and 26 kD in 293T cells,respectively,with a titer of 1.0×109 cell/mL TCID50.The findings revealed that the recombinant adenovirus with the IPNV VP2 gene and VP3 gene was successfully ob-tained,and the virus had a high titer in 293T cells and could be expressed stably.The successful construction of recombinant adenovirus vectors with VP2 and VP3 proteins of IPNV could provide a reference for the development of the vaccine and better prevention of IPNV in Atlantic salmon.
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维普
