CAJ | 학술논문

目的为了提高大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)的产率，对影响工程菌生长、产物表达和产物提纯等因素进行探索。方法设计3种pH值培养基，以菌得率、表达率为指标，观察pH对工程菌生长和表达的影响。以包涵体得率、纯度和产品得率、纯度、活性为指标，观察表达产物提纯各步对产率的影响。用SDS-PAGE薄层扫描法测定包涵体和产品纯度。用TF1细胞MTT法测定产品rhGM-CSF活性。结果 pH7.0培养基最好，菌得率2.22 g/L，表达率为23%，比其它pH值培养基约高1倍。菌体不经冻存处理即进行破菌制备包涵体，得率可提高2.5倍。在rhGM-CSF复性步中，复性液中加入少量PEG 4000，可提高复性率0.8倍。采用优化条件制备大肠杆菌rhGM-CSF，可提高产率约8倍。结论影响大肠杆菌表达rhGM-CSF产率的因素较多，优化培养、表达、纯化各步的条件，均可大幅度提高产率。
목적위료제고대장간균표체적중조인립세포-거서세포집락자격인자(rhGM-CSF)적산솔，대영향공정균생장、산물표체화산물제순등인소진행탐색。방법설계3충pH치배양기，이균득솔、표체솔위지표，관찰pH대공정균생장화표체적영향。이포함체득솔、순도화산품득솔、순도、활성위지표，관찰표체산물제순각보대산솔적영향。용SDS-PAGE박층소묘법측정포함체화산품순도。용TF1세포MTT법측정산품rhGM-CSF활성。결과 pH7.0배양기최호，균득솔2.22 g/L，표체솔위23%，비기타pH치배양기약고1배。균체불경동존처리즉진행파균제비포함체，득솔가제고2.5배。재rhGM-CSF복성보중，복성액중가입소량PEG 4000，가제고복성솔0.8배。채용우화조건제비대장간균rhGM-CSF，가제고산솔약8배。결론영향대장간균표체rhGM-CSF산솔적인소교다，우화배양、표체、순화각보적조건，균가대폭도제고산솔。
Purpose　The aim is to study the influence factors of recombinant E.coli cell growth,product expression and purification,in order to raise the product rate of rhGM-CSF.Methods　Three pH media were planed to observe the effects of pH on E.coli cell growth and expression with the indexes of the cell density and expression level.The influences of various treatments on product rate were observed with the indexes of the volume and purity of inclusion bodies and the yield,purity,activity of product.Using SDS-PAGE method for analysis of purity.The product bioactivity was determined by using TF1 cell and MTT colorimetric method.Results　The cell density in the medium pH 7.0 was 2.22 g/L;the product expression rate was 23%.It was two times higher than other pH media.The inclusion body prepared by bacteria without freezing store was 2.5 times volume than freezing store.During renatura-tion PEG 4000 could raise 0.8 times active product.The optimum conditions used rhGM-CSF product rate could raise 8 times.Concluston　The product rate of rhGM-CSF expressed in E.coli was affected by many factors.The optimum condition for culture,expression and purification could raise quite a number of product rate.