Cloning And Prokaryotic Expression Of Xinjiang Karakul Sheep TNF-α Gene And The Analysis Of Its Antigenicity

Tumor necrosis factor alpha, a cytokine with complex bioactivity,is mainl y secreted by macrophages, monocytes, polymorphonuclear leukocytes, mastcel ls and smooth muscle cells. In recent years, basic and applied research on the TNF gene has gradually become one of the important studies of animal breed ing for disease resistance.In this experiment, This Xinjiang Karakul sheep was takan as the candid ates for the reasch.A pair of primers was designed and synthesized according to the sheep TNF-alpha gene sequence in Genbank, accession Number:NM₀01024860. Coding sequence of TNF-alpha gene was amplified by RT-PCR t echnology from total RNA of Karakul sheep lymphocytes. The TNF-alpha targe t genes was cloned to pMD-18T vector and transfer into competent DH5a bact eria then got the positive recombinant, then recombinant sequence was identifie d by PCR and double digests method. sequenced by Shanghai Sengon biologic al engineering technology and service Co., Ltd.The sequence is1268bp,includin g a705bp an open reading frame which codes234animo acids. TNF-alpha s equence comparison shows that the reference sequence homology of up to99.86%of TNF-alpha domain is more conservative and cattle, dolphins, wild boar, the homology analysis was92.47%,86.99%,87.1%,83.56%. TNF-alpha sequ ence has three potential O-glycosylation sites, twelve potential phosphory sites, Sequence in the presence of71alpha helices, beta43fold zone, and a120r andom coil.Then TNF-a was ligated into pET-28b vector and was identified by PCR, digestion with endonuelease, which proved to constructed a prokaryotic expressi on plasmid pET-28b-TNF-α successfuly,then transformed pET-28b-TNF-α into E. coli BL21and inducd by IPTG. Expressed approximately49KDa fusion prot ein. Optimal expression conditions were determined by SDS-PAGE.The optial induction time was4.0h, the best temperature was37℃, and the optimal co ncentration of IPTG was1.0mmol/L.Under the optial expression conditions, th e fusion protein account21.6%of the total bacterium protein by Bandsean5.0. Western-blotting result. Detect ing pET-28b-TNF-α protein soluble.The results sho w that the prokaryotic expression plasmid pET-28b-TNF-αexisted in form of th e solible protein.Using the purified TNF-α rabbit were immunized45days afte r immunization,Western-blot and the agar diffusion test analysis demonstrated t hat the expressed TNF-αprotein could be detected by positive serum from rabbi t that the expressed pET-28b-TNF-αprotein has good reactivity, immunogenicity.