关键词: 重组角质细胞生长因子-2(rhKGF-2)； 高密度培养； 柱层析, 转受体FGFR2-Ⅲb, BaF3细胞株, 生物活性

Abstract:

We extracted total RNA from human embryonic lung fibroblast cells and obtained cDNA by means of RTPCR. Then the PCR products were inserted into pET3c that was transformed into BL21 (DE3) host cells, which was induced to develop solubility protein products. Highdensity culture was conducted  in fedbatch mode under the control of fermentation conditions. Purification was carried out by cation exchange chromatography and heparin affinity chromatography. And the effect of rhKGF2 on proliferative activity was tested via transferring receptor FGFR2Ⅲb of the BaF3 cells. We constructed gene engineering bacteria of recombinant human keratinocyte growth factor2 (rhKGF2), studied the highdensity culture of recombinant engineering bacteria and purification process and established the method of activity determination. Total protein is more than 30.0% of the total bacterial protein over consecutive three batches of fermentation. The purity of rhKGF2 was higher than 99.0% after twostep column chromatography. We built a new way to test its biological activity. The result shows that it seemed obviously curved letter S, which had some relation with the dose in some extent.

Key words: recombinant human keratinocyte growth factor2(rhKGF2), highdensity fermentation, column chromatography, receptor FGFR2Ⅲb turn, BaF3 cell, biological activity