| Coding sequence (CDS) of mature interferon-τ(blFN-τ) gene from Chinese Hostein was cloned and sequenced for the first time (GenBank Accession Number: DQ680846,DQ680847). The structure and function of blFN-τwere predicted by bioinformatics methods. The results showed:The CDS of mature blFN-τgene was 519bp, encoded blFN-τwhich composed of 172 amino acid residues. The CDS of mature blFN-τgene and correlative sequences from GeneBank fell into four groups by cluster analysis. A novel group (τ4) and six novel variant forms (τle,τ3f,τ3g,τ3h,τ4a,τ4b) were found. DQ680846 was a variant form inτ4, which was designatedτ4a; DQ680847 was a variant form inτ3, which was designatedτ3f. The identities were 92.7%～99.8% in nucleotide sequence among all blFN-τgenes, and that were 88.5%～99.4% in amino acid sequence. The identities were 94.0%～97.1% and 92.9%～99.6% respectively in nucleotide sequence amongτ4a,τ3f and other blFN-τgenes, and that were 91.4%～96.0% and 88.5%～99.4% respectively in amino acid sequence. The IFN-τgenes of different species fell into four groups by cluster analysis: cow, American bison and buffalo comprise one clade; sheep, goat and musk ox comprise another; gayal, red deer and musk deer comprise a third clade; giraffe comprise an entirely different clade. The identities were 84.6%～99.2% in nucleotide sequence among IFN-τgenes of all species, and that were 68.4%～97.7% in amino acid sequence. The identities were 87.7%～99.2% in nucleotide sequence among bIFN-τgeneτ4a,τ3f of Chinese Hostein and IFN-τgenes of other species, and that were 74.1%～97.7% in amino acid sequence. The structure and function of blFN-τwere predicted by bioinformatics methods. One potential N-glycosylation sites in blFN-τamino acid sequence was found, Asn78 could be glycosylated. Five main hydrophobicity region of blFN-τwere found, which stand 53～458, 75～86, 96～104, 114～121 and 139～156 in amino acid sequence respectively. Six potential phosphorylation sites in blFN-τamino acid sequence were found: Ser25, Ser137, Ser153, Ser155, Thr119 and Tyr136. Two disulfide bond of bIFN-τformed between Cysl and Cys99, Cys29 and Cys139 respectively, in order to maintain spatial structure of bIFN-τ. The three dimensional structure of bIFN-τbased on five a-helices. Theseα-helices formed by 111 amino acid residues with amount of 64.53% of bIFN-τsequence; the interval amongα-helices standed by random coil which formed by 61 amino acid residues with amount of 35.47% ofbIFN-τsequence. 3～155 of blFN-τamino acid sequence was typical conserved domain of type I IFN, which consisting of IFN-α,β,ω,τ,δAs a result, we can conjectured that bIFN-τpossess mutual biological function of type I IFN, such as antivirus, anti-proliferation and immunoloregulation.The bIFN-τgene was inserted into prokaryotic expression vector pET-28a(+). Thus we constructed recombinant fusion expression plasmid pET-28a-blFN-τ. Then pET-28a-bIFN-τwas transformed into E. coli BL21(DE3) and expressed with induction of IPTG. The expressed condition was optimized and the recombinant fusion protein was purificated. The results showed:Both the insert direction and reading frame of blFN-τgene at the cloning junction were correct; and blFN-τgene had already expressed in E. coIi BL21(DE3) with amount of 20.68% of the total bacterial proteins. The expression capacity could be increased to 28.84% by optimizing expressed conditions. The expression capacity of fusion protein which was induced at 37℃and 30℃are higher than 25℃and 20℃. The fusion protein expressed in lh after induction and the expression capacity reached peak value in 3h after induction. The recombinant fusion protein was purificated by immobilized metal ion affinity chromatography (IMAC) methods. The purification effect of Elution buffer which contained 300～500mmol/L imidazole are better. Both concentration and purity of the recombinant fusion protein are high after purification. |
