Construction of recombinant adenovirus vector against infectious hematopoietic necrosis and infectious pancreatic necrosis in Oncorhynchus mykiss
Infectious hematopoietic necrosis virus(IHNV)and infectious pancreatic necrosis virus(IPNV)are etiological agents of viral diseases of Oncorhynchus mykiss,and the concurrent infection of the two viruses is very common among modern trout hatcheries,which has caused huge economic losses to farming industry.In this study we optimized a recombinant adenovirus to simultaneously express the G gene and VP2 gene by FMDV 2A peptide in HEK-293 cells to obtain high titers to produce a recombinant adenovirus.cDNA was synthesized from total RNA of IHNV and IPNV and amplified for the target gene using G gene,VP2 gene,and F2A gene primers.These products were further sequenced to confirm the positive DNA products for IHNVG,IPNV F2A and VP2 genes by comparing with their corresponding target sequences.The IHNVG gene,F2A gene and IPNWP2 gene were inserted into a linearized adenovirus shuttle plasmid pAdTrack-CMV vector by using PCR and multi-gene fragment homologous recombination technology.The positive plasmids CMV-G-F2A-VP2 were digested using Not Ⅰ and Hind Ⅲ restriction enzymes.Linearized recombinant vector plasmids were extracted for digestion with Pme I.The recombinant adenovirus shuttle plasmid CMV-G-2A-VP2 was then co-transformed with adenoviral backbone plasmid pAdEasy-1 into electro-competent BJ5183 cells to construct a recombinant adenovirus plasmid Adv-G-F2A-VP2,which were digested using Not Ⅰ and Hind Ⅲ restriction enzymes,and then the positive recombinant adenoviral plasmids were transformed into competent Escherichia coli DH10B cells to obtain high-copy plasmids.Finally,the recombinant adenoviral plasmids Adv-G-F2A-VP2 were digested with Pac Ⅰ and analyzed by agarose gel electrophoresis;the bands corresponding to the target DNA fragments were recovered and then sequenced.The recombination empty plasmids of pAdTrack-CMV empty vector and pAdEasy-1 vector were used as negative controls.The transfected cells were determined by monitoring the expression level of green fluorescence protein(GFP)for 8 d.The cells and medium were collected according to GFP expression level.The cell suspension with viruses was used to inoculate HEK-293 cells again for more recombinant adenovirus particles.The recombinant empty adenovirus of plasmids without IHNVG and IPNWP2 genes were prepared and used as negative controls.The expression of glycoprotein and VP2 protein was confirmed by western blot,and the recombinant viral titer was detected and calculated.The result suggested that the IHNVG,F2A and IPNWP2 genes were cloned with the gene length of 3 036 bp.These results clearly demonstrated that the recombinant plamid CMV-G-F2A-VP2 was successfully constructed.In addition,the recombinant plasmid Adv-G-F2A-VP2 was confirmed by enzymatic digestion with Pac I.As anticipated,an approximately 34.5-kb DNA fragment and a smaller band with the size of about 4.5 kb were observed,suggesting that IHNVG and IPNVVP2 genes were successfully inserted into the recombinant adenovirus plasmid by homologous recombination.Two extra proteins were expressed in HEK-293 cells at 48 h after transfection with the recombinant adenoviral plasmid.The larger protein with a molecular mass of apptoximately 58 kDa was detected,and the smaller one with a molecular mass of apptoximately 54 kDa was tested.This result indicated that the recombinant adenovirus with IHNV G and IPNV VP2 proteins might be successfully packaged in HEK-293 cells.Based on fluorescence intensity and distribution of GFP,the infection attack rate and severity of recombinant adenovirus on HEK-293 cells were evaluated.GFP was observed in approximately 60％of the total HEK-293 cells after transfection with the recombinant adenovirus,where the titer of recombinant adenovirus was calculated and it was 1 × 109 5 mL-1 TCID50.The finding revealed that the recombinant adenovirus with IHNVG gene and IPNVVP2 gene was successfully obtained,and the virus had a high titer in HEK-293 cells and could be expressed stably.This study offers foundation for developing a bivalent recombinant adenovirus vaccine with IHNV G and IPNVVP2 genes and provides new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile Oncorhynchus mykiss.
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