Study On Phylogeny Of VP2 Gene Of Feline Panleukopenia Virus And The Immunogenicity Of Recombinant Proteins

Feline panleukopenia is a highly contagious viral disease of kittens with a highmortality caused by feline panleukopenia virus (FPV), a member of parvoviridae. Notonly can FPV infect all members of the cat family , but also other wild animals aresusceptible to infection, such as raccoons, coatimundis, and ringtails et al, which has posea serious threat to the economic animals and wildlife. The serological survey onantibodies against FPV showed that infection of FPV occurs worldwide, and there werehigh positive rate in the sera of wild animals in China. In order to comprehensivelyunderstand the infection situation of FPV in northeast tigers, 184 sera samples weredetected by HI test. PCR and virus isolation techniques were used to detect FPV in fecalsamples of tigers. The results showed that 49.5% (92/184) serum samples were HIpositive, which indicated that the infection of FPV is common in northeast tigers. 9 in 24fecal samples were PCR positive, out of which 7 strains of FPV were successfullyisolated by F81 cell line. To clarify the genetic evolution of epidemic strains, a pair ofspecific primers was designed and used to amplify VP2 genes of 7 isolates. Then VP2genes were cloned into T vector for sequencing. The full length of the VP2 gene of 7isolates is 1755 bp, which encodes 584 amino acids, with the same lengths as the VP2gene of standard strain CU-4 of FPV. The homology of nucleotide and amino acidsequences among 7 domestic isolates were 98.5-100.0% and 98.9-100.0%, respectively.phylogenic tree based on VP2 genes showed that different strains of FPV can cluster intotwo genogroups. The genogroup I includes some strains isolated from foreign countries,such as d, PLI-IV, philips Roxane, 23 and CU-4 , while the genogroup II include mostdomestic strains isolated in China as SM-4, GT-2, GT-3, JF-1, JF-3, ZF-5, HT-69 and 377.Although there were some variation in VP2 gene, most variations were synonymousmutation rather than the missense mutation. This study clarifies the molecularcharacteristics of genetic variation of domestic strains of FPV.Vaccination is an effective measure to prevent feline panleukopenia. In order todevelop subunit vaccine of FPV, the expression of VP2 gene of FPV in Pichia pastorisyeast was carried out in the study. The VP2 gene fragment encoding major antigenicregions of VP2 protein was amplified from strain GT-2 by PCR and cloned into pGEM-Tvector, generating pTVP2. And the purified EcoRI/NotI VP2 fragment was subcloned intoyeast expressing vector pPICZαA , generating pPICZαA VP2. Then the recombinantpPICZαAVP2 was linearized with SacI and transformed into competent GS115 forexpression. The positive yeasts were screened by PCR. The expression of recombinantVP2 protein was identified by SDS-PAGE and Western-blotting. Under induction of 1%methanol, a protein with molecular weight of 32 kDa was successfully expressed by therecombinant yeast, which was specifically recognized by polyclonal antibody againstFPV. The recombinant protein amounts for 24.8% in the total protein of supernatant bygel scanning analysis. After three times of immunization with the recombinant VP2protein in mice, the neutralizing antibodies and HI antibodies in sera reached 1:16 to 1:32and 1:64 to 1:128, respectively, which indicated that recombinant VP2 protein was ofbetter immunogenicity.Recombinant canine adenovirus type 2 (CAV-2) expressing the VP2 protein of FPVwas successfully constructed. Firstly, complete genome of CAV-2 was extracted and thendigested by Kpn I. The 4.9Kb Kpn I fragment containing E3 region was recovered andcloned into pVAX1, generating pVAXE3. The 859bp fragment in E3 region of pVAXE3was deleted by the digestion of SspI, generating pVAX△E3. Secondly, the purifiedNdeI/SphI expression cassette fragment harboring VP2 gene form pVAXVP2 wassubcloned into the shuttle vector pVAX△E3, generating pVAX△E3VP2. Thirdly, thepurified SalI/NruI expression cassette fragment from pVAX△E3VP2 was further clonedinto the backbone vector pPoly2-CAV2 containing complete genome of CAV-2,generating pCAV-2-FPV-VP2. To gain the recombinant adenovirus, the plasmidpCAV-2-FPV-VP2 was linearized by PvuI/AscI to release recombinant genome, and thentransfected into MDCK cell through lipofectamine. The recombinant virus CAV-2-VP2was gained through 4 times of passage in MDCK, and the typical cytopathic effects (CPE)of CAV-2 could be observed. 3.1Kb gene fragment contain VP2 gene of FPV can beamplified from the genome of recombinant virus. The specific mRNA was also detectedby RT-PCR. 70 kDa recombinant protein VP2 expressed by recombinant virus could bespecifically recognized by polyclonal serum against FPV in western blot analysis. ThePCR detection showed that the recombinant virus displays better genetic stability through20 passages. After three times of immunization with the recombinant virus CAV-2-VP2 inmice, the FPV neutralizing antibodies and CAV-2 HI antibodies in sera reached 1:16 to1:32 and 1:64 to 1:128, respectively, which indicated that VP2 protein expressed byrecombinant virus CAV-2-VP2 can effectively induce the specific humoral immunizationagainst FPV.Based on the results of immunization in mice, twenty-five seronegative kittens wereimmunized with recombinant VP2 protein and recombinant virus CAV-2-VP2,individually or co-immunized by intramuscular injection. After three times ofimmunization, the specific antibodies against FPV were detected by HI, ELISA and SNtest, while specific cellular immunoresponse were assayed by lymphocyte transformationtest. The results of ELISA and SN test shows that the groups immunized by recombinantVP2 protein, recombinant virus CAV-2-VP2 and co-immunization can all efficientlyinduce the specific cellular and humoral immunoresponse. Meanwhile, the groupimmunized by recombinant virus CAV-2-VP2 and the co-immunization group couldinduce the similar antibody titers, which was higher than the group immunized byrecombinant VP2 protein but lower than the positive control immunized by FPV weakvirulent strain. The results also revealed that the recombinant virus CAV-2-VP2 caneffectively induce not only the specific antibody against FPV, but also the antibodyagainst CAV-2. The results of challenge experiments showed that immunization byrecombinant virus and co-immunization of recombinant VP2 protein and recombinantvirus could develop better immune protection. This study laid foundation for thedevelopment of subunit vaccine and recombinant live viral vector vaccine of FPV.