Duck Alpha Interferon Original (true) Expression And Biological Activity

In this paper,the study is on Sichuan Province local varieties of duck interferon. We conducted the following series of studies:Sichuan duck typeⅠinterferon IFN-αORF gene sequence was cloned,identified and analysis;IFN-αORF prokaryotic gene expression vector and eukaryotic expression vector were successful constructed; interferon-αhas been expressed by prokaryotic expression &.biological activity;Useing ducklings as model,by means of immunohistochemistry.We studied on pcDNA-SDIFN-αvector dynamic distribution of expression in ducks;At the same time as the immunoadjuvant,we test the pcDNA-SDIFN-αeukaryotic expression vector to carry on immunological regulation function in ducks.The results were as follows:1.Sichuan duck IFN-αORF gene cloning,sequencing,analysis and protein structure prediction.Application software Oligo6.0,designed two pairs of specific primers within BamHI and EcoRI restriction sites,by PCR and nest-PCR method,we get two size amplified from duck liver geome about completely duck interferon-αgene ORF DNA fragment,Small fragments of which is more suitable for expression.Using pGEM-T vector,we get clone about duck interferon-αgene.Using ClustalX sequence analysis software,as well as with beijing ducks,chickens,geese,and other sequences in the NCBI comparison,The results showed that Sichuan and Beijing duck ORF sequences are very similar,its nucleotide homology to 99.13%,amino acid homology of 98.96%.And chicken nucleotide and amino acid homology are 71.8%and 49.97-50.47%,the goose nucleotide homology of is 96%.At the same time using BioEdit,NetNGlyc,TreeView software speculate that the gene protein was analyzed.The results show that Sichuan duck has two potential glycosylation sites,a protein kinase C phosphorylation sites,and a casein kinaseⅡphosphorylation sites,the signal peptide cleavage 28-29(ANA and FS) between amino acids,and mature duck interferon-αhas five hydrophobic,no transmembrane region.2.SDIFN-αORF prokaryotic gene expression vector pBV220-α-SDIFN Construction, expression and the anti-viral activity detection.BamHI and EcoRt restriction,by the end of visco-directional link,prokaryotic expression vector pBV220-SDIFN-αplasmid was constructed.And the plasmid was transformed into prokaryotic expression strain Ecoli DH5α,using temperature-induced and SDS-PAGE.The results showed that product is 40 KD.By purified and refolding and VSV /DEF system,we detected the anti-viral activity of product.The results of antiviral activity are about 150 U / ml,vitality about 440 U / mg.Application FQ-PCR method,dynamic monitoring of the virus nucleic acid about DHBV in vivo and DPV in vitro.The results showed that the interferon has a strong inhibition of virus amplify.3.SDIFN-αORF eukaryotic expression vector pcDNA-SDIFN-αconstruction and identification.By means of primers with ends of the EcoRI &.BamHI restriction sites,we get gene from the pGEM-T vector,and link with the pUC 18 directional positions.And then use EcoRI &.Xbal double digestion of eukaryotic expression-vector pcDNA3.1(+) and pUC 18-SDIFN-α,we get pcDNA-SDIFN-αeukaryotic expression vector clone.By BamHI, EcoRI and XbaI three digestions and sequencing shows that it is correct about Sichuan duck IFN-αeukaryotic expression recombinant pcDNA-SDIFN-αplasmid.4.Immunohistochemistry's Established and reasearch on eukaryotic expression plasmid pcDNA-SDIFN-αexpression laws in duck.Using pcDNA-SDIFN-αeukaryotic vector immune duck,the 16 different duck organizations were collected at the different time.We established the immunohistochemistry method.And application the method,we tested the expression laws of interferon in vivo.pcDNA-SDIFN-αimmune duck,continued to express time up to nine weeks.In the lung cells,heart muscle cells,liver cells and spleen cells,intestinal glands and intestinal villi cells,muscle cells,skin cells successfully expressed,14 d-35d in a peak of expression.Thymus,pancreas,Bursa and Bemhard's glands were not detected yellow particles.The earliest site is in Lung &.immune tissue.Brain tissue's Positive reaction is almost no change with times.By Gene gun immunization method,the content of exprcssion shpws of Group 6μg＞3μg group＞1μg group.Intramuscular immunization ducks,the content of expression shows of 200μg group＞100μg group＞50μg group, the all of which have a dose related.Gene gun immunization method is earlier than intramuscular gene in expression;3 h can be detected expression products by Gene gun immunization method.At the same time,comparing to intramuscular injection method,the gene gun use less plasmids.5.The study on Eukaryotic expression plasmid pcDNA-SDIFN-αas immune adjuvant.pcDNA- SDIFN-αeukaryotic expression vector as for immune adjuvant,research on the DPV / DVH attenuated vaccine regulation of immune activity.At different times,by MTT assay determination of T lymphocytes,the number of CD3 + T lymphocytes and specific changes in the IgG antibody levels about DPV immuned duck.The results show that a certain dose of pcDNA-SDIFN-αeukaryotic expression vector as an immune adjuvants have positive role in regulating.