Construction And Immune Character Of Fusion Protein Of Ubiquitin And GP5 Glycoprotein Gene Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV), a causative agent of highly contagious disease with worldwide distribution, can cause severe economic losses to the swine industry. The GP5 is one of the major factors of the virus to which the host responses well with protective antibody production. The recombinant plasmids containing the GP5 gene can induce specific immune responses against PRRSV. The ubiquitin-proteasome pathway is an efficient pathway for protein degradation. It's mainly responsible for the selective degradation of many protins in eukaryotic cells.The Ub fusion vaccines were designed to accelerate turnover of transgene antigen and increase the variety and number of peptides available for major histocompatibility complex (MHC) binding.Two pairs of specific primers which have the restriction enzyme site BglⅡ, NotⅠ respectively were designed. About 620bp PCR product was obtained from pcDNA3-ORF5 plasmid that had been constructed with PCR. The product of PCR and pCMV-Ub vector were digested by the restriction enzymes, and purified, respectively, and ligased. The ligation was transformed into E.coli strain DH5 a competent cells and selected with ampicillin. Positive clones were picked up on the LB plate with ampicillin after 12h and then inoculated into LB culture. A small scale of plasmid was extracted and identified by restriction enzymes and sequencing. The results showed that the GP5 gene was cloned into pCMV in the correct open reading frame, and they were named pCMV-Ub-GP5 and pCMV-GP5 respectively. The nucleotide homogeneity between GP5 of S1 strain and other ioalated strain was analyzed in the NCBI with Blast biosoft, and the homogeneity was about 99%.The efficacy of PRRSV GP5 DNA vaccines between pCMV-Ub-GP5 and pCMV-GP5 were compared. Mice in each group were immunized with 100μg of plasmid byintra-dermal injection. All groups were immunized four times in all with frequency of 15 days. The results showed that the humoral responses were both induced in group pCMV-Ub-GP5 and pCMV-GP5. The level of ELISA antibody and neutralizing antibody was equivalent between group pCMV-Ub-GP5 and group pCMV-GP5. On time of 15d after the third immunization, the OD490 of ELISA antibody of both groups reached 1.0 and neutralizing antibody could be detected. On time of 15d after the fourth immunization, neutralizing antibody of group CMV-Ub-GP5 reached 7 and group CMV-GP5 reached 12.In conclusion, the present study demonstrated that the recombinant plasmid of ubiquitin-conjugated GP5 were constructed and the plasmid induced humoral response in mice, as well as the recombinant plasmid with GP5 gene only. The cellular immunity of recombinant plasmids should be studied furtherly.