Construction Of Recombinant Adenovirus With Encephalomyocarditis VP1Gene And Its Immune Effect Research On Mice

Encephalomyocarditis virus (EMCV) is a newly discovered virus, one can infect a variety of animals and people with myocarditis, encephalitis and myocardial inflammation. This virus has caused great harm to human life and health, but its biological characteristics and pathogenic mechanism is unclear because of its late discovered, so there isn’t have efficient diagnosis and prevention materials.So this research analyzes the VP1protein spatial structure and properties with the help of bioinformatics analysis method. The VP1gene was cloned with EMCV RNA by RT-PCR, and construct recombinant adenovirus vector pAd-VP1-EGFP, which was then packaged in HEK293cells to get recombinant adenovirus rAd-VP1-EGFP. The CHO-K1cells were infected with recombinant adenovirus to detect VP1protein expression and location. Mice after immuned with rAd-VP1-EGFP were infected with EMCV to immune effect of recombinant adenovirus. This research was to provide technical support for EMCV live vector vaccine.The results showed that1. VP1protein is composed of277amino acids, and belonged to hydrophilic amino acid, with three strong hydrophilic areas located in100,150and200amino acids, which were located on the surface of protein. Among the seven predicted linear epitopes, the three strong hydrophilic areas was included, and also has potential transmembrane domain. VP1homology is high. Results showed that VP1has strong conservation, and most of its areas as were hydrophilic, can be candidate for genetic engineering vaccine preparation.2. The recombinant adenovirus vector pAd-VP1-EGFP was successful constructed, and packaging in HEK293cells, the constructed recombinant adenovirus TCID50was10-7.5/0.1mL3. The recombinant adenovirus virus can infect CHO-K1cells without CPE, and can detect VP1gene overexpression by RT-PCR. Western-blotting and IFA results indicated that VP1protein mainly located on the cell membrane and has good immunogenicity. The results showed that recombinant adenovirus can infect CHO-K1in vitro, and VP1gene can be expressed in the cell normally, which can lay the foundation for further eukaryotic virus antigen expression research. 4. Immune mice with recombinant adenovirus, the results indicated that after the seond immunization, it produced obvious higher specific antibodies. The twice immuned mice protection researched93.3%, significantly higher than40%of single immune mice. It showed that the recombinant adenovirus vaccine has good prospects in the prevention of EMCV.In a word, through this research, the recombinant adenovirus was successful constructed, with good immunity effect in mice, and lay the foundation for further EMCV genetic engineering vaccine development.