Construction And Optimization Of Gene Expression Vector Of The Bacillus Subtilis HemA And HemL

5-aminolevulinic acid(ALA) is the biosynthetic of the tetrapyrroles, such as porphyrin, Heme, chlorophyll (CHL), and vitaminB12.The ability of ALA to generate photosensitization derivatives in human bodies, insects and certain plant species make it possible to be used as an antitumor medicine, herbicide, insecticide, as well as growth promoting factor and stress tolerance improver for plants. In December 1999 ALA was authorized by FDA as a photodynamic medicine to treat skin cancer in prophase. And it is drawing more and more attentions of scientists to apply it in the treatment of various epidermis cancers. Recently, ALA has been applied to the treatment of acne vulgaris and the suppression of the inflammatory response to coronary and iliac injuries. As the second generation of photodynamic medicine, its outstanding advantages include less side effect, better curative effect and penetrability, lower price, etc. As an agricultural chemical, ALA at least has the advantage of being biodegradable and having no accumulation in environment.In this study,the hemA gene and hemL gene were amplified through polymerase chain reaction from B.subtilis genome DNA, and construction the expression vector pGJ216 and pGJ218 of their genes by Escherichia coil-Bacillus subtilis which was used to shuttle vector. In order to gain the High-level expressed in B.subtilis 1A747 cell, the two vectors were electric transformation when induced by maltose. The target proteins of HemA and HemL were highly expressed the content of it up to 11 % and 13 % respectively in total soluble protein through SDS-PAGE analysis. The determination by 5-aminolevulinic acid showed that the level of 5-aminolevulinic acid were 26.31 mg/L and 24.47 mg/L in fermentation supernatant of recombinant bacteria of pGJ216 and pGJ218. In addition, the color of bacterial liquid was red, the colourated substances was proved that was porphyry substance by ultraviolet spectrophotometers. It showed that recombinant proteins had the physiological activity and promoted the synthesis of the 5-aminolevulinic acid, and accelerated the catabolism ways in the same time.Construction expression vector pGJ216M and pGJ218M was to optimize, the promoter–PglvM of mutation Pglv was respectively cloning pGJ216 and pGJ218 hemA and hemL gene upstream, and replace the Pglv promoter, and expression vector of pGJ216M and pGJ218M was obtained in this course. Its electric transformation to the competent cells of B.subtilis 1A747 and induction expression with the maltose. SDS-PAGE analysis showed that expression protein of pGJ216M and pGJ218M take on an obvious protein strip near the 50KD, but the treatment of control bacteria had no clear protein strip. The results demonstrate that the construction of expression vector pGJ216M and pGJ218 Mwas successfully and achieved highly expression in the Bacillus subtilis. And then, the biological anabolism was improved by the expression of 5-aminolevulinic acid, the synthesis speed of 5-aminolevulinic acid was accelerate strongly.