重组人KGF制备工艺和活性检测方法的建立

Preparation of Recombinant Human Keratinocyte Growth Factor and Its Biological Activity Study

目的:在毕赤酵母中实现了KGF的高效表达,并初步建立了生物学活性检测方法。方法:合成5’端缺失69个核苷酸的KGF基因序列,克隆入pPIC9并转化毕赤酵母菌株GS115中,经诱导表达。发酵液上清采用脱盐层析和阳离子交换层析进行分离纯化。利用貂肺上皮细胞(Mv-1-Lu)检测其生物学活性。结果:表达水平达到了110mg/L发酵液;表达产物经一步离子交换层析就能得到有效分离,总收率在50mg/L发酵液以上;纯化的rhKGF生物学活性与KepivanceTM相当。结论:rhKGF制备工艺和检测方法的建立将为该因子的规模化生产和进一步的临床应用提供良好基础。

Objective： High level and industrial preparation of recombinant rhKGF was achieved in Pichia pastoris. Method： The KGF coding sequence lacking the 23 N - terminal amino acid residues from the origin sequence of KGF （FGF7） in the Genebank was synthesized chemically, cloned into the pPIC9K vector and integrated into Pichia pastoris strain GS115. The rhKGF expression was induced by using methanol. The rhKGF was purified from the supernatant of culture by desalination and cation exchange column chromatography. MTY method with mink lung epithelial （ My - 1 - Lu） cells was used for the determination of its biological activity. Result： Efficiently rhKGF was expressed in Pichia pastoris and purified by cation exchange column chromatography, At least 50rag of interested protein was obtained from 1L Media. The purified protein has the similar biological activities to KepivanceTM launched in the market. Conclusion： The established methods of preparation and biological activity studies for KGF will provide fundamental base for its large - scale production and further anDlications in clinical studies.