Expression And Immunogenicity Research Of Rabbit Hemorrhagic Disease Virus In The System Of Recombinant Adenovirus

Rabbit hemorrhagic disease(RHD) is a highly contagious and fatal disease caused by Rabbit hemorrhagic disease virus(RHDV).The capsid protein(VP60) is the exclusive structure protein and can make animals produce neutralised antybody and directly related to immune response. Now the widely used vaccine of RHDV is a kind of inactived vaccine, although it has a long good immune effect, but it has its own inevitable deficiencies. The cell culture in vitro method of rabbit hemorrhagic disease virus has not yet been established., Researcher take the focus on the develop alternation approaches for producing vaccines in recent years,such as gene engeering vaccine. The research amplified capsid protein (VP60) gene of rabbit hemorrhagic disease virus by reverse trancript polymerase chain reaction (RT-PCR) technique and cloned the fragment into the system of adenovirus expression to constract recombinant adenovirus, then expressed capsid protein in HEK293 cell. The immunoprotection effect of the expressed VP60 protein had been detected. This should be useful to reserch gene engineering vaccine of rabbit hemorrhagic disease virus (RHDV).According to German reference strain of GeneBank database of gene sequences (M67473), a pair of VP60 gene-specific primers are synthesized, Kpn I restriction sites and start codon inserted in the upstream of the primer's 5'extrem , and Xho I restriction sites and stop codon inserted in the five downstream of the primer's 5'extrem. 1740 bp RT-PCR product was amplified from Shanghai rabbit liver, and was cloned into PMD19-T vector, then transformed into DH5αand selected with the ampicillin. Extracted plasmid of the positive bacteria was identified with Kpn I and Xho I, and the target fragment was sequenced. DNAStar software analysis showed that the size of amplified fragment was same as expectations and fully consistent with vector open reading frame. The homology was from 90.2% to 98.2% between this VP60 gene sequence (named SH-VP60) with the reference sequence isolated from other domestic and international.Target gene was Subcloned into pShuttle-CMV vector, and the recombinant plasmid pShuttle-VP60 was transformed into host bacteria E.coli DH5α. Homologous recombination between linearized pShuttle-VP60 plasmid and pAdEasy-1vector occurred in E.coli BJ5183 after electrotransformation. Consequencetly, the VP60 gene fragment was inserted into pAdEasy-1 vector through selecting with kanamycin and the recombinant plasmid pAd-VP60 was received. pAd-VP60 plasmid was linearized by restrictive endonuclease Pac I and was transfected into HEK293 cell with LipofectamineTM2000. The recombinant adenovirus was harvested when the CPE is obvious.The expression of VP60 was identified by the IFA, SDS-PAGE and Western-blotting. The result showed that adenovirus VP60 protein (about 60 KDa) has expressed in HEK293 cells.In this study more than 2-month-old non-immunized RHD rabbit was immunized with the expressed RHDV VP60 protein. The results showed that the expressed recombinant protein can induce anti-RHDV in rabbits after 22 days of immunization, the anti-RHDV titers in serum range from 24 to 27. when challenged with more than 210 HA unit of lethiferous RHDV, the immunized animals survived for immunity protection provided by antibody.