Genetically Engineered Downstream Standardization System Technology

The production of genetic engineering product is a very complicated systematic projects, and can be divided into two stages in the upper reaches and the lower reaches. The upper reaches technology including separate the target DNA and construct the engineering bacteria is the indispensable foundation of developing genetic engineering product; and the output and the quality of the genetic engineering product is being directed control by the lower reaches technology. This subject uses the genetic engineering technology and the newest modern biotechnology to study the genetic engineering lower reaches stage, in order to research and establish the optimum, high density fermentation technology pattern and high efficient isolation and purification model of recombinant proteins from inclusion bodies.Section- I : To research and establish a kind of high density fermentation technology pattern.What the question that people are concerned about most chiefly was plasmid stability keeps , the high efficiency of target DNA is expressed and the influence to the purification in lower reaches. According to these problems, we adopt to the method of mending material, optimize to fermentation media and partly ferment condition. Finally, we excogitate a kind of fermentation technology that is suitable for target gene efficiency expressed and is advantageous of product purified. With the plasmid pBV220-IFNr, pBV220-HGFa, pBV220-HGFb,pBV220-hPK5 that expresses serve as the model, adopting the BIOSTAT-C15L of B. Braun company, utilize the method of mending material to ferment, through optimization fermentation media and optimization partly ferment condition (ventilate quantity, stir speed, mend material speed), eventually establishment a kind of fermentation technology that is suitable for target gene efficiency expressed and is advantageous of product purified. Fermentation density OD600=40-42, SDS-PAGE and Densitometric scan analysis, the result show that expression level is 20% of total bacterial proteins.SectionII :To research and establish a kind of high efficient purification model of recombinant proteins produced in Escherichia coli as inclusion bodies.To raise separate efficiency and biological active efficiency is especially important in the purification model of recombinant proteins. In accordance with these questions, we raise inclusion bodies purity before the purification as far as possible, and select the suitable chromatogram technology. With the model of IFNr, HGFa HGFb, hPk5, study its washing conditions and purification technology. After washing with reagent, adopt the newest purification technology SOURCE30RPC , SDS-PAGE and Densitometric scan analysis, the result show that expression level is 90% of total bacterial proteins. After renaturation, IFNr, HGFa, HGFb,hPK5 were purified by AKTA purifier chromatogram instrument,Sepharose Fast Flow,Ssphacrayl series gel, selecting optimize condition. Finally establish a kind of high efficient purification model of recombinant proteins produced in Escherichia coli as inclusion bodies , purification productpurity >98%.Conclusion:We have established a kind of high density fermentation technology pattern and established a kind of high efficient purification model of recombinant proteins produced in Escherichia coli as inclusion bodies. At present, producing recombinant proteins with genetic engineering technology have gotten extensive development in our country, this program explored out a practical feasible procedure for the lower reaches of genetic engineering fermentation and purification technology.