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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: To achieve high expression of KGF in Pichia pastoris and establish a preliminary biological activity assay. Methods: KGF gene sequence with 69 nucleotides deleted at 5 ’end was synthesized and cloned into pPIC9 and transformed into Pichia pastoris strain GS115. The fermentation broth supernatant was purified by desalting chromatography and cation exchange chromatography. Its biological activity was detected using mink lung epithelial cells (Mv-1-Lu). Results: The expression level reached 110mg / L fermentation broth. The expressed product could be effectively separated by one-step ion-exchange chromatography with the total yield above 50mg / L. The biological activity of purified rhKGF was comparable to that of KepivanceTM. Conclusion: The establishment of rhKGF preparation and detection methods will provide a good foundation for the large-scale production and further clinical application of this factor.