Molecule Clone And Prokaryotic Expression Of Bovine Interferon-tau Gene Mature Protein Coden Region And It's Biological Function In Pregnancy Establishment

Bovine interferon-tau(bIFN-Ï„) is from trophoblast of early embryo and it interrupted the process of antiluteolytic mechanism in bovine endometria,promoted corpus luteum developing,and finally caused the beginning of gestation.Many studies have suggest that bIFN-Ï„was crucial role in pregnancy recognition and it regulated the transition from pregnancy recognition to embryo implantation,bIFN-Ï„was a member of IFN-I family,it has the common character of IFN-I,such as antivirus,anti-proliferative activity and immuno-modulation but bIFN-Ï„only expressed in embryonic trophoblastic cells without virus induction condition.The cytotoxicity of bIFN-Ï„was lower than other I-IFN.Based on the above-mentioned biological functions and features of the IFN-Ï„,the studies on IFN-Ï„have gradually become hot in the current.In our studies,materials are Chinese Holstein cow.The code sequences of bIFN-Ï„mature protein were gained by bioinformatics analysis.Firstly,the sequences were cloned with pMD18-T vector in E.coli JM109 and sequenced,bIFN-Ï„fusion protein with histidine tag were expressed in engineering bacteria E.coli BL21(DE3) with pET28a(+) vector.Homology,genetic distance and molecular evolution relationship of different subtype bIFN-Ï„,IFN-Ï„from different species and the member of bovine IFN family were analyzed by molecular genetic evolution.Secondly,the basic physical parameter, glycosylation site,phosphorylation site,kinase action site,hydrophobicity,disulfide bond position,secondary structure,tertiary structure,conserved structure domain were predicted by proteome analysis tools.The difference between bIFN-Ï„expression product and bIFN-Ï„mature protein were elucidated by prediction.Thirdly,bIFN-Ï„expression products were purified with affinity chromatograph and a whole process for bIFN-Ï„expression product purification was established.Finally,cultured bovine endometrium cells were as a model in vitro.Under in vivo hormone concentration condition,after cultured endometrium cells being treated with bIFN-Ï„,progesterone,bIFN-Ï„and progesterone,expression level of some genes related with pregnancy recognition and embryonic implantation were analyzed.The main results of the studies were as follow:Two DNA sequences were identified from four clones which were from different individual.Squencing results shown:the CDS sequence size of bIFN-Ï„mature protein from Chinese Holstein cow was 519 bp,only an exon in the whole open reading frame. The sequence coded bIFN-Ï„mature protein including 172 amino acids.The two-sequence was submitted to GenBank.After being accepted the accession numbers were DQ680846 and DQ680847.In the different subtype bIFN-Ï„,nucleotide sequence homology ranged from 93.1%to 99.8%,and amino acid sequence homology ranged from 87.9%to 98.8% by nucleotide sequence deduction.In different species,nucleotide sequence homology ranged from 84.2%to 99.2%,and amino acid sequence homology ranged from 67.1%to 97.1%by nucleotide sequence deduction.The results indicated bIFN-Ï„homology was very high and it was conserved gene in evolution.Different subtype bIFN-Ï„were divided into 3 groups by cluster analysis.DQ680846 was clustered intoÏ„4(Ï„4a),DQ680847 was clustered intoÏ„3(Ï„3f);Different species IFN-Ï„were analyzed by clustering.The results showed bIFNÏ„3f and bIFNÏ„-4a were on a branch,and the relationship was closer with America bison in phylogenesis.The results of bIFN clustering supported the conclussion;bIFN-Ï„4a and bIFN-Ï„3f were belonged to I-IFN.In I-IFN family,the relationship was closer bIFN-Ï„with bIFN-δand bIFN-ωthan with bIFN-αand bIFN-β.Recombinated vector with bIFN-Ï„(pET-28a-bIFN-Ï„) were sequenced.The results showed insert-way of bIFN-Ï„gene and read-frame was correct.The difference between bIFN-Ï„expression product and bIFN-Ï„mature protein were analyzed by proteome analysis tools.The results indicated:glycosylization site,disulfide bond position,tertiary structure and conserved structure domain was no difference.Compared with dissolvability and isoelectric point of bIFN-Ï„mature protein,these of bIFN-Ï„expression products increase,their half life prolongation and more stability.Between bIFN-Ï„mature protein and bIFN-Ï„expression product,the difference of kinase action site was a little. bIFN-Ï„expression product added a phosphorylation site on N-19 Ser,added random coil structure of histidine tag on N-terminal.After E.coli(with pET-28a-bIFN-Ï„) being cultured 4 hours under 37℃with 1.0mmol/L IPTG induction,expression fusion protein reached to 28.84%in total protein from E.coli BL21(DE3).Inclusion body was purified with spinning after induced E.coli being washed and ultrasonic disrupt.The yield of inclusion body reached to 2.6983±0.1967g per liter E.coli cultured media.Inclusion body was denatured with denatured buffer solution including 8mol/L urea and bIFN-Ï„expression product was purified with HisTrap HP affinity column.In elution condition,imidazole elution was significantly better than pH-griaent buffer solution(P＜0.05).The elution buffer solution including 400mmol/L imidazole was significantly better than others(P＜0.05).Under the best elution condition,Purified ratio of bIFN-Ï„expression was the highest,it reached to 26.36%in total protein which was loaded on column.Denatured expression product was refolded with chromatograph.The results indicated:urea concentration was decreased from 8mol/L to 1.5mol/L in elution buffer solution and bIFN-Ï„expression product was eluted from affinity column too. Electroconductivity of elution solution including bIFN-Ï„expression product obviously decreased,from 50mS/cm to 4mS/cm,by HisTrap Desalting column.Isoionic point of purified bIFN-Ï„expression products were tested by isoionic focusing electrophoresis. After purified bIFN-Ï„expression product were cleaved with o-iodosobenzonic acid their peptide finger-print were analyzed.The results indicated:high purified bIFN-Ï„expression products were included in elution solution and affinity chromatography is ideal method for bIFN-Ï„purification.Cultured endometrium cells in vitro could grow in DMEM-Ham's F₁₂ medium which comprised 0.2nmol/L estrogen,100 IU/mL Penicillin,100μg/mL streptomycin, 2μmol/mL Glu and serum or serum-free.Cells type in primordial and passage generation were endothelial-like cells which were from endometrium endothelial cells,and fibroblast-like cells which from endometrium stroma-cell. Effects of bIFN-Ï„and progesterone on expression level of bovine endometrial genes related with pregnancy recognition and embryonic implantation were analyzed by fluorescent quantitative PCR.The results indicated:progesterone significantly suppressed the expression of estrogen receptor-alpha(ER-α),cycloxygenase-2(COX-2) and tissue inhibitory metalloproteinases-2(TIMP-2) in bovine endometrium(P＜0.05) and significantly promoted the expression of estrogen receptor-beta(ER-β),matrix metalloproteinases-2(MMP-2) in bovine endometrium(P＜0.05).Progesterone promoted and stabilized the expression of progesterone receptor(PGR),didn't effect the expression of oxytocin receptor(OXT-R),progesterone receptor membrane component-1(PGRMC1) and ubiquitin cross-reactive protein(UCRP) in bovine endometrium,bIFN-Ï„significantly suppressed the expression of ER-α,ER-β,OXT-R and TIMP-2(P＜0.05) and promoted the expression of COX-2,prostaglandin E synthase(PTGES),PGR and UCRP in bovine endometrium(P＜0.05).bIFN-Ï„could stabilized the expression of MMP-2.PGRMC1 expression was up-regulated under progesterone and bIFN-Ï„synergistic action condition.Effects of treatment time on expression level of bovine endometrial genes related with pregnancy recognition and embryonic implantation were analyzed.The results indicate:Progesterone regulated gens expression slowly and bIFN-Ï„regulated genes expression quickly.In PGRMC1,OXTR,TIMP-2,COX-2,PTGES,PGR,ERα,ERβ, UCRP and MMP-2 ten genes,PGRMC1 and OXTR rapidly reflected to regulative signal, then were TIMP-2,COX-2,PTGES.PGR,ERα,ERβ,UCRP and MMP-2 slowly reflected to regulative signal.