| Diabetes mellitus is a dysbolism disease lacked relatively or absolutely insulin itself. To sustain their lives,a part of patients of diabetes need injection of exogenous insulin,which is extracted from animal pancreas chiefly. As a result of the amino-acid difference between human insulin and animal insulin, the latter has side effect and bring about anguish and inconvenient to the patients.With the rapid development of molecular biology and gene technology, it become a hot pot to produce efficient human insulin and its analogue by using the method of biotechnology. During the procedure of using E.coli to produce human insulin, target protein usually forms inclusion body so that we perform denaturation and renaturation to obtain active human insulin,which not only brings about inconvenience to separation and purification, but also affects final yield of target protein. Therefore,we hopes to express proinsulin gene with solution and build efficient process of purification through logical design,expression system selection and optimum fermentation condition in the study.PET32-a vector and E.coli BL21expression system was choosed in the study. We obtain human proinsulin gene by connecting synthetic fragment and PCR. Firstly, we linked proinsulin gene with PMD 18-T vector and screened right recombinant vector. then we inserted right proinsulin gene into pET32-a vector to get expression vector . Proinsulin gene was expressed together with TrxA·tag and His·tag in the host , E. coli strain BL21.By the experiment of fermentation condition study and induction strategy, we obtain the optimum condition of 300C culture temperature and 0.6mM IPTG induce density. SDS-PAGE and Westem blotting analysis indicate that expressed protein has the same molecular weight with theoretic one,and has the immune response with insulin antibody, which proves proinsulin gene is expressed rightly.Separation and purification were studied after fermentation in order to achieve purified mature human insulin. The broth was centrifuged and collected E.coli. After separation by IMAC, ion-exchange chromatography was employed to purify the target protein. Purified heterologous protein was digested by enterokinase and carboxypeptidase B. IMAC was used secondly to wipe off TrxA and His·Tag.At last,gel chromatography was performed to separate human insulin and linking peptide.The first eluate peak we collected is human insulin sample.Enzyme-linked immunosorbent assay analysis indicated that the heterologous protein sample has a certain bioactivity of human insulin. |
