Construction And Expression Of Recombinant Adenovirus Co-expressing The GP5 And M Protein Of Porcine Reproduction And Respiratory Syndrome Virus In Coat

The swine’s immune system is a double-edged sword for porcine reproductive and respiratory syndrome virus(PRRSV) infection.On the one hand PRRSV has a predilection for immune cells,because of PRRSV-specific binding of immune cells, notably macrophages, PRRSV appears to replicate extensively,if not exclusively,in cells of the immune lineage,the direct replication of which may lead to immunosuppression, precipitate secondary infection and/or mediate disease,which can cause a variety of infectious disease vaccine immunity failed.On the other hand,the virus stimulates immunity post-infection that protects an animal from re-infection.At present, because of the unsatisfactory effect and safety of PRRSV vaccine,people keep trying to study a new generation of vaccine.In the prevention of PRRS,national and international experts reported that the point of the development of effective prevention of PRRS vaccine is nucleic acid vaccine. GP5 and M proteins are the star proteins of nucleic acid vaccine because of their specific antigen characteristics.Some studies were found that the fusion of M and GP5 proteins expressed by recombinant vector could produce a better immune effect than individual. Fusion expression of GP5 and M proteins become a new directions of the development of more efficient prevention PRRSV vaccine. Currently,the recombinant adenovirus is one of the most efficient vector systems for the delivery of vaccine antigens.Adenovirus vector has been widely used in the human gene therapy,and developed a variety of human and animal genetic engineering vaccine.The mammary gland has been identified as an attractive option for the production of eukaryotic modified recombinant proteins.The heterologous proteins synthesized in the mammary epithelial cells immediately secrete to milk, and these proteins can be purified by relatively simple chromatography systems.We use dual promoter of adenovirus expression system to select the PRRSV structural protein M and GP5 tandem fusion expression,so as to better play PRRSV M,GP5 protein specific antigen activity and synergistic effect,and verify the initial expression of these proteins in goat mammary epithelial cells and breast milk, provides a new way for the study of gene engineering vaccine of PRRSV.The main contents and results of this experiment are as follows:(1) Successfully cloned 1221 bp in the N terminal with signal peptide and his tag expressing GP5 and M protein fusion connection CDS,construct the expression with a signal peptide, his tag in GP5 and M fusion connection protein of recombinant adenovirus vector p BHG-Signal P-His-GP5-M. RT-PCR verified the vector was successfully constructed.(2)Two step bacterial homologous recombination was used to successfully pack the adenovirus r HAd-Signal P-His-GP5-M,and the amplification of the virus titer measured obtaining 1011 TU / ml.(3)Packaging adenovirus r HAd-Signal P-His-GP5-M successfully infected mammary epithelial cells,and the expression of GP5-M gene was detected by Q-PCR in mammary epithelial cells infected with virus.The expression of GP5-M protein in the mammary epithelium and cell supernatant was detected by Western blotting.(4)Packaging adenovirus r HAd-Signal P-His-GP5-M pour into the goat mammary gland in vivo,and the highest expression of GP5-M fusion protein in the collected milk was detected by Western blotting.