提高bFGF基因表达水平的策略及其表达产物的纯化工艺

目的通过在同一个表达载体pLX1上调整SD序列和ATG之间距离，以提高bFGF目的蛋白表达量。方法以Klenow和MungBeanNuclease通过增加2个及减少2个碱基调整SD序列和ATG之间距离，SDS-PAGE和Westernblot检测目的蛋白表达量，以高压液相疏水层析、凝胶过滤及肝素亲和层析纯化目的蛋白、MTT法进行活性测定。结果获得了重组质粒pLX2，pLX3，诱导后与表达质粒pLX1（7.78%）相比，表达量分别为8.03%，9.9%，经纯化后获得的bFGF纯度达98%，ED50为2.29ng/ml。结论调整SD序列和ATG之间距离可以增加目的基因的剂量,从而提高了目的基因在大肠杆菌中的表达水平。

Strategy on Enhancement of bFGF Gene Expression and Purification of Its Products

Objective: To adjust the distance between SD sequence and ATG in the same expressive plasmid pLX1 to enhance expression of heterologous bFGF gene in E. coli. Methods: Adjusting the different distance between SD sequence and ATG by Klenow and Mung Bean Nuclease. SDS PAGE and Western blot showed the expressed protein bFGF in E.coli. bFGF proteins were purified by HPHIC, HPGFC and HAC. Biological activity was examined by MTT. Results: Recombinant plasmids pLX2, pLX3 were obtained and the expressive levels were 8.03%, 9.9% respectively. Also the purified bFGF was obtained by HPHIC, HPGFC, HAC and its ED 50 was 2.29 ng/ml. Conclusion: Increasing the bFGF gene dosage by adjusting the distance between SD sequence and ATG could increase the expression level of a desired protein.