| Orf virus(OrfV), a member of Parapoxvirus, Poxviridae family, which contain an envelop and linear double stranded DNA. OrfV is a pathogeny of which is characterized by papules, empyesis, ulceration and scabs on the skin of the lips, tongues, nose, and breast or the mucosa. Orf is also a contagious,addicted to the epithelial and animal infectious diseases. Orf occured worldwide and epidemic, it's a viral diease which threaten to the sheep industry and has economic significance. Vaccination is a reliable and effective method in prevention and control of viral epidemic. The frequently-used vaccines included the attenuated virus, inactivated vaccines. But the scabby vaccine exists the risk of scattering virus, and the attenuated vaccine exists reversion of virulence and other defects.So developing the effective genetically engineered vaccine has become an inevitable trend.Interleukin-2(IL-2) can be used as a cytokine immune adjuvant in combination with nucleic acid vaccine, which can enhance immunity. This study constructed IL-2 gene eukaryotic expression vector and B2 L, F1 L gene eukaryotic expression vector of OrfV.Then combined the cytokine adjuvants with OrfV eukaryotic expression vector to immune mice to explore the IL-2 cytokine adjuvant effect of immune effect of DNA vaccine of OrfV.1.Identification and construction the eukaryotic expression plasmid of caprine IL-2 gene: collected the peripheral blood collected from healthy Guizhou black goat,isolated and cultured lymphocytes, then acquired the IL-2 gene through RT-PCR amplification and sequencing, and analyzed the IL-2 gene with bioinformatics softwares;The pVAX1 vector was been used as eukaryotic expression vector to construct IL-2 gene eukaryotic expression plasmid, identified by plasmid PCR, double digestion and sequencing methods. Then transfected the recombinant plasmid into MDBK cells,and identified it's expression by RT-PCR and ELISA. The results showed that Guizhou blackgoat IL-2 gene encoded a complete reading frame of 468 bp, encoding 155 amino acids.Compared with caprine IL-2 sequence in GenBank, they shared the a nucleotide consistency of 95.9%-100%, and amino acid consistency of 91.1%-100%. Then the caprine IL-2 gene was submitted to GenBank, and received the accession number was KT934548. Bioinformatics prediction results showed that the IL-2 protein had more?-helical and ?-fold, a signal peptide, no transmembrane domains and specific features, the performance of the entire peptide chain is hydrophilic. The PCR, double digestion and sequencing results of the recombinant plasmid showed the construction of pVAX1-IL-2plasimd was successful. The RT-PCR and ELISA results showed that there were mRNA of IL-2 transcribed in the supernatant of MDBK cells transfected by pVAX1-IL-2 after 48 h,and the IL-2 protein concentration was about 112 pg/mL in the supernatants. This study constructed pVAX1-IL-2 eukaryotic expression plasmid and expressed in the MDBK cells successfully.2.Identification and construction of p VAX1-B2 L, pVAX1-F1 L eukaryotic expression plasmid of OrfV: according to the OrfV gene sequences in GenBank(No.AY386264), the B2 L and F1 L gene primers was designed respectively. The B2 L and F1 L gene of OrfV-GZ strain was amplified, cloned and sequedced by PCR, and analyzed by bioinformatic softwares. The pVAX1 vector was been used as eukaryotic expression vector to construct B2 L and F1 L genes eukaryotic expression plasmid, identified by plasmid PCR, double digestion and sequencing methods. Then transfected the recombinant plasmid into MDBK cells,and identified it's expression by RT-PCR and IFA. The resulted showed the the length of B2 L gene was 1137 bp,the F1 L gene was 1029 bp. Compared with 14 strains in GenBank, the length of B2 L gene from different strains were the same,they shared the consistency of nucleotide were among 97.3%-98.8%, the consistency of amino acid were among 97.1%-99.7%.. While the length of F1 L gene were varies, the consistency of nucleotide were among 96.0%-99.6%, the consistency of amino acid were among 95.5%-99.7%. Then the B2 L and F1 L genes of OrfV-GZ strain were submitted to GenBank respectively, and received the accession numbers KP994595 and KP057582.Bioinformatics prediction results showed that the B2 L protein had more?-helical and?-fold, the entire peptide chain is hydrophobic, the protein no signal peptide or transmembrane domain but 2 conserved domain of phospholipases D(PLD) superfamily,included the specific binding sites. The F1 L protein also had more?-helical and ?-fold, the entire peptide chain is hydrophobic, the F1 L protein had two transmembrane domains but no signal peptide. The PCR, double digestion and sequencing results of the recombinant plasmid showed the construction of pVAX1-B2 Land pVAX1-F1 L plasimds was successful.The RT-PCR and IFA results showed there were mRNA of B2 L and F1 L gene transcribed in MDBK cell, the specific-green fluorescence was detected by IFA in MDBK cells. These results illustrated the pVAX1-B2 Land pVAX1-F1 L plasmids could be expressed in MDBK cells.3.Effects of IL-2 gene adjuvant on orf virus DNA vaccine immune effects: the mice were immunized by pVAX1-B2 L, pVAX1-F1 L, and pVAX1-B2L+pVAX1-F1 L combined with pVAX1-IL-2 as the adjuvant, respectively. The ELISA method was adopted to detect the specific antibody of OrfV and cytokines of Th1 type(IL-2, IFN gamma and TNF-?) and Th2 type(IL-4, IL-6, IL-10). The MTT method was adopted to detect the proliferation of spleen lymphocyte of mice. The FACS method was adopted to detect the subgroup of spleen lymphocyte. The results indicated that the three groups of pVAX1-B2 L, pVAX1-F1 L and pVAX1-B2L+pVAX1-F1 L showed they had good immunogenicity. Specifict anti-OrfV antibodies were induced in all immunized groups at the first week after the immunization. The antibodies reached the highest level at the sixth week, then dropped at the seventh week, the pVAX1-B2L+pVAX1-F1L+pVAX1-IL-2group performed an obvious trend. MTT method showed that the lymphocyte proliferation of three groups were higher than pVAX1 and PBS groups, the difference was extremely significant(P<0.01). These groups added pVAX1-IL-2 adjuvant compared with groups which without pVAX1-IL-2, the difference was extremely significant(P<0.01). These results indicated that the pVAX1-IL-2 showed immune enhancement contribution in the three groups, the lymphocyte could be induced a continual proliferation at the first to the fourth weeks. FACS results indicated that the content of CD3+CD4+ T-lymphocyte of pVAX1-B2 L, pVAX1-F1 L, pVAX1-B2L+pVAX1-F1 L, pVAX1-B2L+pVAX1-IL-2,pVAX1-F1L+pVAX1-IL-2, and pVAX1-B2L+pVAX1-F1L+pVAX1-IL-2 groups were higher than pVAX1 and PBS groups, and the pVAX1-B2L+pVAX1-F1L+pVAX1-IL-2 was the highest among all immune groups. The content of CD3+CD4+ T-lymphocyte of these groups added pVAX1-IL-2 were higher than the groups which without pVAX1-IL-2. The content of CD4+ T-lymphocyte of all groups was higher than CD8+ T-lymphocyte, and the specific value of CD3+CD4+/CD3+CD8+ T-lymphocyte was between 1.71~2.94. These results indicated that all the plasmids could induce the proliferation of the lymphocyte's subgroup, the helper T lymphocytes was higher than inhibitory T lymphocytes, and all the plasmids could not cause adverse reactions in mice. The cytokines results showed that the content of Th1 type(IL-2, IFN-?, TNF-?) was higher than the Th2 type(IL-4, IL6,IL-10), and these groups added the pVAX1-IL-2 were higher than the groups which not added pVAX1-IL-2, these results indicated the Th1 type immune response occupied the main position in the cellular immunity.Conclusion:The study cloned the IL-2 gene of black goat of Guizhou, B2 L gene and F1 L gene of OrfV-GZ strain, respectively and successfully. The pVAX1-IL-2, pVAX1-B2 Land pVAX1-F1 L eukaryotic expression plasmids were constructed successfully. The pVAX-IL-2 as the cytokine adjuvant could promote the specific humoral immunity and cellular immunity of pVAX1-B2 L, pVAX1-F1 L and pVAX1-B2L+pVAX1-F1 L in mice, and the Th1 type immune response occupied the main position. |
PDF Full Text Request