The effects of induction tem perature on porcine interferon-α (pFN-α) production, cell m etabolic activity and transcriptional level of sam key enzymes in m ethanolm etabolic pathw was investigated w ith a recombr iant Pihia pastoris as m ode strain . The fed -batch cultivation w as in plan ented in a 5 L bbreactor, and he transcrip tional level of som e key enzym es in stable induction phase w as quan tiffed by ReatiinePCR technobgy. R esulls show ed lhatcompaied to taditbnal30°C induction, a bw induction temperature of 20℃ could significantly in prove pFN-α antiviral activily, cell metabolk activily and 1he tianssrptbnal bvels of key enzym es in m eihanol m etaboli paihw ay. The m axin urn pFN-α antiviral activily at 20Κ roached 5 .57×106 IU/mL , whih was 19 .2 fold of the m axim um when inducing a t30℃ . The enhanced pFN-α antiviral activily ob tained at20℃ w as assrbed to:(1) the higher m ethanol consum ptbn rate and cell m etabolic activily; (2 ) the strengihened energy generation and detoxiffed pathway, and m ore eneigy ibr cell constituen ts ; (3 ) the formaldehyde assim ilatory pathw ay w as activated significantly and m ore carbon flux tow ards cell constituen ts and pro tein expression.%以一株表达猪-α干扰素(pIFN-α)重组毕赤酵母为研究对象,考察了诱导温度对pIFN-α表达、细胞代谢活性及甲醇代谢关键酶的影响.在5 L发酵罐开展毕赤酵母高密度流加培养生产表达pIFN-α研究,利用实时PCR技术对不同诱导温度、诱导稳定期的胞内甲醇代谢关键酶(包括解毒酶)基因转录水平进行定量分析.低温(20℃)可以同时提高细胞的代谢活性和猪-α干扰素的表达水平,低温(20℃)下最高pIFN-α抗病毒活性达到5.57×106 IU/mL,是30℃常温诱导下最高抗病毒活性(2.9×105 IU/mL)的19.2倍;相比于30℃ 常温诱导,20℃下醇氧化酶(AOX)、甲醛脱氢酶(FLD)、甲酸脱氢酶(FDH)、过氧化氢酶(CAT)及二羟基丙酮合成酶(DHAS)基因的转录水平明显提高,其中CAT和DHAS转录水平的增加尤为显著.低温诱导提高pIFN-α表达原因在于:(1)甲醇利用速度快,细胞代谢活性强;(2)低温诱导下甲醛产能途径和解毒能力得到加强,为合成细胞物质提供更多的能量;(3)甲醛同化途径关键酶(DHAS)转录水平在低温下增强更为显著,促使更多的碳流向细胞合成和外源蛋白表达.
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