Study On Rabies DNA Vaccine And Recombinant Live CAV-2 Vaccine Based On The Glycoprotein And Nucleoprotein CDNAs Of Strain ERA

The glycoprotein (RGP) and the nucleoprotein (NP) are the two main components of the five structure proteins of rabies virus(RV) , which can induce protective immune response after inoculation of animals. To develop new vaccines against rabies virus infection, the following molecular biology experiments have been carried out . The cDNAs of RGP and NP genes of strain ERA of RV were amplified by RT-PCR,cloned, sequenced and analysed. The RGP and NP genes of strain ERA were subcloned into the eukaryotic expression plasmid pVAX1,named pVG and pVN,respectively. The mice and dogs were immunized with pVG and pVN by intramuscular injection. RV recombinant live CAV-2 vector vaccines were successfully constructed. The RGP expression cassettes were ligated into the E3 deletion region of canine adenovirus type 2 (CAV-2), named CAV-2/rgp. The dogs were and immunized with the recombinant vaccines. Cloning and analysis of the RGP and NP cDNAs of RV strain ERA: Two pairs of primers were designed and synthesized according to the published cDNA sequences of RGP and NP. Reverse transcription polymerase chain reaction (RT-PCR) was employed to amplify the full length of cDNAs of RGP and NP. Sequencing results showed that the RGP cDNA was 1575 bp in length and included an open reading frame, which encodes a 524 amino acids polypeptide. Compared to strains PV, CVS and SRV9 , the nucleotide homology was 98%,89% and 99%;and amino acid homology was 96%,88% and 99%,respectively. Antigenic index analysis revealed that the index of the RGP of strain ERA is as good as or higher than that of PV, CVS and SRV9. The full length of the NP cDNA is 1353 bp, encoding a 450 amino acids polypeptide. When compared to strains PV, CVS and SRV9 , the nucleotide homology was 99%,93% and 99%;and amino acid homology was 98 %,98% and 98%,respectively, demonstrating that the NP is highly stable and conservative among different strains. Antigenic index analysis revealed that the immunogenicity of the NP of strain ERA is as good as or higher than that of PV, CVS and SRV9. Construction of the eukaryotic expression plasmids pVG and pVN and expression of the RGP and NP by transient transfection into Vero cells: Recombinant plasmids pVG and pVN were constructed by inserting the RGP and NP genes into pVAX1, respectively. Vero cells were transfected with the recombinant plasmid by lipofectamin. The cell lysates were assayed by ELISA for antigen expression 72-96h post transfection. The results showed that both the RGP and NP were expressed in Vero, and which were further confirmed by specific reactivity to the RV antisera . Preparation in a large scale of the pVG and pVN and immunizing animal with them: The pVG and pVN were prepared in a large scale by a molecular biology method. The plasmids was dissolved in TE to form 50ug/100ul DNA vaccine . Four groups of mice were immunized with a dose of 100uL of plasmid pVAX1,pVG,pVN and human inactivated rabies vaccine, respectively.Three groups of dogs were immunised with a dose of 400uL of plasmid pVAX1,pVG,pVN,respectively. The mice and dogs were immunised for three times at 14 day intervals. Indirect ELISA was employed to determine the immune response. SN antibody of immunized dogs induced with pVG was assayed. The results showed that specific antibodies occurred in all groups after three times of immunization ,except for negative controls. Construction and identification of recombinant live CAV-2 vector vaccine based on the RGP cDNAs of strain ERA: The pVG gene was conducted, site-directed mutation by molecular biology technology. The pVG expression cassette was ligated into the E3 deletion region of canine adenovirus type 2 (CAV-2). RV recombinant vaccines were constructed. The RGP expression cassettes was released from pVG by digestion of MluI +BbeI , blunting and ligation into SspI site of pVAX?E3 . The recombinant plasmids were named pVAX?E3rgp. The orientation of the expression cassette is consistent with the transcript orientation of adenovirus E3 region . pVAX?E3rgp and pPoly2-CAV-2 were digested with NruI+SalI, respectively. The purified DNA fragment containing the...