Cloning And Fusion Expression In Escherichia Coli Of Thanatin Gene

OBJECTIVE:To chemcally synthesize the thanatin gene and induce its expression in escherichia coli as gluthathion S-transferase(GST)-fusion protein. Isolated snd purified the product. Underlaying the further study of the antibacterial activity and the application value of thanatin.METHODS:1.Obtained the gene According to the sequence of amino acid which reported by reference. the gene of thanatin with preferred codons of E.coli containing the cleavage sites of restriction enzymes EcoR I and Sal I was chemically synthesized.2.Constructed cloning recombinant plasmid Digested the gene and pUC18 plasmid by EcoRⅠand SalⅠ, ligated them and transformed into E.coli DH5a competence. Through a-complementary, the positive recombinant was identified and sequenced.3.Constructed expressing recombinant plasmid Digested the recombinant and pGEX-4T-1 with the same restriction endonucleases and ligated them.And then transformed into E.coli BL21. The positive recombinant was identified and sequenced.4. Expressed and purified The positive recombinant was induced by IPTG at 37℃for 2-5 hours.The product GST fusion protein was observed by SDS-PAGE of thallus protein. Harvested the positive expression E.coli BL21.According to GST Mini SpinPurification kit, schizolysised the inclusion bodies and purified GST fusion protein. Finally GST fusion protein was enzymolysised by thrombin.RESULTS:1.Two single strands of Thanatin gene phoring EcoRⅠand SalⅠwere chemically synthesized and renaturated to double strands.2.Thanatin gene was cloned into pUC18 vector, then checked with sequence analysis. The result coincided the designed gene sequence indicating the success of constructing cloning recombinant plasmid.3.The fusion expressing vector of thanatin was constructed successfully. Sequencing analysis indicated that thanatin gene was correctly inserted into the vector. So it could be induced expression.4.Induced expression under IPTG for 3 hours, the fusion protein band about 29kD appeared on SDS-PAGE gel indicating that thanatin gene had been expressed. Schizolysised the inclusion bodies and purified,solved GST fusion protein and enzymolysised by thrombin. Acquired the solution including thanatin protein.CONCLUSION: Thanatin gene was transformed into E.coli BL21. Under the IPTG induction acquired the GST fusion protein. Then enzymolysised with thrombin. Acquired the solution containing thanatin protein. The further purification was needed to obtain thanatin protein with higher purity.