Study On Subunit Vaccine Recombined By CSFV Specific T Cell Epitope Antigen And Porcine Parvovirus-Like Particles

Procine parvovirus(PPV) is one of the major etiological agent of reproductive failure in pigs. It belongs to the family Parvoviridae. Pigs of all ages are susceptible to the virus, The clinical signs of PPV are characterized by abortion ,fetal abnormality, mummification and aciesis. Classical Swine Fever Virus(CSFV)causes acute and highly contagious viral diease in pigs, All kinds of ages pigs are sensitive to the virus. The typically clinical manifestation of Classical Swine Fever(CSF) is blood-poisoning. Abort, lymphocytes decrease and secondary infection and other signals are also happened in partical infected pigs. Now.CSF has become one of the severe viral diseases that lead to a severe economic loss in pig farming, So, It is essential to develop a safe and effective vaccine to prevent the disease in China. In this study, we made the VP2 capsid protein of Procine parvovirus as a virus carrier; the peptide E290(a CSFV specific helper T-cell epitope and a CTL epitope existed in this peptide, which corresponds to amino acid residues 1446-1460 of the CSFV non-structural protein NS2-3, as the carried gene to develop vaccine for PPV and CSF preventing. This idea of vaccine development has never been reported. Procine parvovirus LJL12 isolate was propagated on PK-15 monolayer cell. A pair of oligonucletide primer was designed to amplify the gene coding for the VP2 protein of PPV, The primers were chosen by the analysis of the reported strains sequences available in GenBank.VP2 gene was amplified by PCR from LJL12 DNA. The product of PCR is approximate 1.7Kb in length. The VP2 gene was cloned into pMD–18T vector, and identified by restriction endonuclease, PCR and sequencing. The result showed that the VP2 gene has cloned successfully. The VP2 gene sequence was analyzed by homology coparing with other published VP2 gene in GenBank. The homology of VP2 gene complete sequence shares more than 99% in nucleotide and more than 98% in amino acid with other PPVs. The result shows that VP2 gene is highly conservative. VP2 was inserted in XhoⅠand KpnⅠmultiple cloning sites of the vector pMelBacA and named PM-VP2. PM-VP2 was identified and analyzed by corresponding restriction endonuclease and PCR on the base of the genetic sites of pMelBacA, Which was identified as VP2 gene of PPV. According to the sequence of CSFV T cell epitope gene reported by Brown. A pair of primers was designed to amplify E290.The sense primer has eighteen complementarity base with antisense primer, The product was cloned into the PM-VP2 recombinant vector, PM-VP2-E290 was identified and analyzed by corresponding restriction endonuclease and sequencing. The result indicated that amplified E290 gene was 60bp in length and shared 100% homology with the designed sequence. Purified PM-VP2-E290 and Bac-N-Blue DNA were cotransinfected insect cell sf9 and harvested recombinant baculovirus 4d later. Identification with PCR showed hybrid E290-VP2 gene has been recombinant correctly with baculovirus DNA and named P-1. P-1 was infected on monolayer sf9 cell after the third plaque purification to reproduce in quantity, and named P-2. The P-3 was made as P-2. After P-3 was infected on sf9 monolayer cell for four days, harvested the CPE cell. The analysis results of SDS-PAGE, Western-blot and Dot-ELISA showed that PPV VP2 gene was expressed in baculovirus. expression product was approximate 67KD. The expression product was observed by Immuno-electron microscopy(IEM).A large number of parvovirus like particles was observed. This result showed that the expressed protein has assembled into particles and the insertion of E290 epitope in VP2 N-terminal was not intact its self-assembl. The BALB/c mice injected with the expressed protein, in the absence of adjuvant. showed that the hybrid recombinant parvovirus like-particles formed by the self-assembly of the VP2 capsid protein of PPV carring the CSFV epitope at its N terminal, not only induced a strong CD8+ class restricted CTL response, But stimulate a highly PPV specific antibody levels.