Isolation And Identification Of Canine Parvovirus Jilin Strains And Cloning And Expression Of VP2-IL-2 Fusion Gene

In recent years,Canine Parvovirus Disease(CPVD)has become an infectious disease that seriously threatens dogs' health,and due to its high mutation rate,the emergence of new strains has been a challenge to the original vaccine immunization prevention means.Research on the new genetic engineering subunit vaccine has become one of the hotspots.In order to improve the immunoprotective effect of the CPV vaccine,this study constructed a recombinant fusion plasmid with Canine IL-2(as the molecular adjuvant)and canine parvovirus VP2 gene.The recombinant protein was expressed by the E.coli prokaryotic expression system.First,12 suspected strains were isolated from dog feces infected with suspected canine parvovirus,and were identified as canine parvovirus through cell seperation,PCR specific fragments amplification,hemagglutination test,TCID50 determination and physical and chemical properties test.Do sequence analysis to the VP2 gene of the obtained 12 CPV strains,they turned out to be five strains of New CPV-2a,four strains of New CPV-2b and three strains of CPV-2c,which indicated that 2a and 2b are the main epidemic subtypes of canine parvovirus in Jilin province.The nucleotide sequence and the deduced amino acid sequence homology analysis showed that among the 12 strains of CPV,the nucleotide homology was 98.9%~99.9%,and the amino acid homology was 99.0%~100%;and when compared with the domestic and foreign strains in GenBank,the nucleotide homology was 98.7%~100% and the amino acid homology was 98.5%~100%.In this study,the isolated CPV-2c JL-11 strain was used as the research object.The CPV-2c type has been evolving and propagating since it was seperated in our country in 2010,and whether the existing vaccine strains have preferable immunologic defence function toward it remains unknown.Therefore,the development of a new genetic engineering subunit vaccine for CPV-2c has a certain clinical value.Then,in primer design,restriction enzyme cutting sites BamH ? and Sac ? were introduced into the upstream and downstream primers to amplify the VP2 gene of CPV JL-11,and the sequence size was 1770 bp.The lymphocytes were isolated from peripheral blood of adult healthy dogs and induced by ConA.The total RNA of the sensitized lymphocytes was extracted and the specific primers were designed.The restriction site Sac? and a piece of flexible linker were introduced in the upstream,and the restriction site Xho I was introduced and in the downstream,so that it was expanded into the coding sequence of the canine IL-2 gene and the sequence size was 501 bp.Homology comparison was done with BLAST between the known canine IL-2 sequences from GenBank and the obtained one,among which the reported canine IL-2 sequence shared the highest homology,up to 99%~100%.Finally,the VP2 gene and the canine IL-2 gene sequence were respectively ligated with the their expression vector pET-28 a after the double-enzyme digestion.The results showed that the recombinant fusion plasmid pET-VP2-IL-2 was successfully constructed.After the recombinant fusion plasmid pET-VP2-IL-2 was transferred into Rosetta(DE3),IPTG was used as the inducer and the optimal induction conditions were established through determination of the optimal induction time and concentration.SDS-PAGE analysis showed successful expression of the fusion protein,which has a molecular weight of about 82 kDa.Western-Blot assay indicated that the recombinant fusion protein had good antigencity.This study provides a basis for the development of a new CPV vaccine.