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当前位置: 首页 > 文章 > 小牛凝乳酶原基因在乳酸克鲁维酵母中的表达及遗传稳定性研究 食品科学 2008,29 (7) 297-302
Position: Home > Articles > Study on Expression of Prochymosin in Kluyveromyces lactis and Genetic Stability FOOD SCIENCE 2008,29 (7) 297-302

小牛凝乳酶原基因在乳酸克鲁维酵母中的表达及遗传稳定性研究

作  者：
冯镇;张兰威
单  位：
东北农业大学食品学院;哈尔滨工业大学食品科学与工程学院
关键词：
乳酸克鲁维酵母;凝乳酶;分泌表达
摘  要：
目的:构建小牛凝乳酶原乳酸克鲁维酵母分泌型表达载体,表达重组凝乳酶原。方法:通过PCR获得小牛凝乳酶原cDNA片段,将目的基因插入酵母表达载体pKLAC1α结合因子分泌信号下游,得到重组载体pKLAC1-prochymosin。SacⅡ线性化后氯化锂转化乳酸克鲁维酵母GG799,用含5mmol/L乙酰胺的YCB固体培养基筛选转化酵母菌,PCR鉴定目的基因,利用特异性引物筛选多拷贝转化子。阳性转化子经摇瓶表达,取上清TCA沉淀后做TricineSDS-PAGE分析并检测凝乳酶的活力,通过检测阳性转化子产凝乳酶的活力考察其遗传稳定性。结果:经测序及PCR证实,凝乳酶原cDNA准确插入酵母表达载体pKLAC1中,转化后重组载体通过同源重组整合进入酵母基因组中。TricineSDS-PAGE分析证明凝乳酶的分子量约为36kD,酸处理后测得培养基中凝乳酶的酶活为83SU/ml,阳性转化子遗传稳定性好。结论:成功构建出酵母表达载体pKLAC1-prochymosin,在培养基中获得分泌的重组凝乳酶原,经过酸处理后凝乳酶原转化为有活性的凝乳酶。
译  名：
Study on Expression of Prochymosin in Kluyveromyces lactis and Genetic Stability
作  者：
FENG Zhen1,ZHANG Lan-wei2,* (1.College of Food Science, Northeast Agricultural University, Harbin 150030, China; 2. College of Food Science and Technology, Harbin Institute of Technology, Harbin 150090, China)
关键词：
Kluyvermyces lactis;chymosin;secreted expression
摘  要：
Objective:To construct Kluyvermyces lactis secreted expression vector and express recombinant prochymosin. Methods :The prochymosin gene encoding mature peptide was amplified by polymerase chain reaction, and then inserted into the downstream of the alpha-mating factor signal of the Kluyvermyces lactis expression vector pKLAC1. Recombinant plasmid pKLAC1-prochymosin was linearized by SacⅡand transformed into Kluyvermyces lactis strain GG799 with lithium chloride. Positive clones were chosen by YCB plates containing 5 mmol/L acetamide and the presence of insert was identified using PCR. Multi-copy integration can be detected using specifical integration primers. The positive transformants were fermented in flask and the proteins in the culture supernatant were deposited with TCA and assayed with Tricine SDS-PAGE. Results: It was proved that the fragment amplified is inserted into the Kluyvermyces lactis expression vector pKLAC1 correctly by PCR and gene sequencing. After lithium chloride transformation, the recombinant plasmid is integrated into the regions of homology within yeast genome. Chymosin activity of the medium is 83 U/ml after acid treatment. Tricine SDS-PAGE analysis proved that the molecular weight of chymosin is about 36 kD. Conclusion: Prochymosin Kluyvermyces lactis expression vector is successfully constructed and recombinant prochymosin is expressed.

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