Abstract:

Abstract:Objective To construct the  recombinant expression vector pET32a-CP6 and  transform  into the Escherichia colic （E.coli） BL21 (DE3) and express the recombinant protein at a high level,in order to provide reliable basis for obtaining a large number of soluble collagen. Methods The recombinant plasmid pET32a-CP6 was transformed into E.coli BL21 and induced by IPTG.The expression conditions of the recombinant protein were optimized in this test,which included temperature,time for induction,concentration of ITPG,inoculation volume,adding time of IPTG.The recombinant E.coli was fermented under the optimal condition,and the lysate of bacteria was purified by nickel ion affinity.The purified target protein was determined for purity by SDS-PAGE.Results The optimal temperature and time for induction of recombinant E.coli were 37℃ and 5 h respectively,while the optimal concentration of IPTG as an inducer was 0.5 mmol·L^-1.The expression level of target protein reached 31.52 mg·L^-1 under the optimal condition.The Western blotting results showed that the recombinant protein had specific reaction with anti-human COL6A2 antibody.Conclusion The optimized recombinant E.coli　can efficiently express the recombinant human collagen,the recombinant protein is purified successfully.

Key words: recombinant human collagen peptide；purification of recombinant protein；Escherichia coli