Construction Of PPV DNA Vaccine PCDNA3.1(+)-VP2-CpG And Elementary Analyses Of Its Immunological Activity

The disease of porcine parvovirus(PPV) is caused by porcine parvovirus which is a major cause of reproduction failure in swine.Parvovirus infections in pigs may cause fetal death and mummification,still births and other reproductive failures in pregnant sows,and dermatitis and diarrhea in piglets.Since PPV was isolated by Carwright in 1967,the virus and it's antibody were proved to be positive in many countries,and the antibody positive rate of PPV reaches to 50%-80%.In china,the antibody positive rate of PPV was up to 80%,which causes lots of economical losses in swine production.The best method to control this kind of disease is prevention.Among the methods,developing vaccine is a key step.DNA vaccine with incomparable advantage is the third generation vaccine which folows pathogeny vaccine and subunit vaccine.In this study,a pair of primers was designed according to SR-1 strain of PPV from GenBank and the main antigen region of VP2 of SR-1 strain was amplified by PCR.And the sequence was 1146 bp,which encoded 382 AA.Analysis of the antigen epitopes of VP2 showed that there are some antigen epitopes located in the region.Concerned this we constructed a eukaryotic expression plasmid pCDNA3.1(+)-VP2 using the eukaryotic expression vector of pCDNA3.1(+),and at the same time,a synthetic oligodeoxynucleotide (CpG-ODN) containing CpG-ISS was cloned into the eukaryotic expression plasmid pCDNA3.1(+)-VP2 to construct another eukaryotic expression plasmid pCDNA3.1(+)-VP2 -CpG.Then the recombinant expression vectors were transfected into COS-7 cells respectively and made them express in vitro.The results showed that the two plasmid also can express VP2 protein.To investigate the immune response of DNA vaccine and the adjuvant effect of CpG-ISS, the recombinate plasmids pCDNA3.1(+)-VP2 and pCDNA3.1(+)-VP2- CpG were largely extracted and were used to immunize the KunMing mice by injecting.Mice were divided into four groups randomly:pCDNA3.1(+) control plasmid group(100μg/mouse);pCDNA3.1(+) -VP2 plasmid group(100μg/mouse);pCDNA3.1(+) -VP2- CpG plasmid group(100μg/mouse) and the blank controller.And at the 0,2,4,5,6 weeks,the PPV antibodies can be detected in serum of all animals inoculated with recombinant plasmids pCDNA3.1(+)-VP2 and pCDN -A3.1(+)-VP2- CpG.And the pCDNA3.1(+) -VP2- CpG plasmid can induce higher titler of PPV specific antibodies.So,the results shows that it is an effective route to enhance the efficacy of DNA vaccine by inserting exogenous CpG DNA into the plasmid.This might be benefit for further research on the safe and efficient PPV DNA vaccine.