| 在毕赤酵母中表达人溶菌酶蛋白的研究 |
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| 引用本文： | 胡乔,赵凌侠,唐克轩.在毕赤酵母中表达人溶菌酶蛋白的研究[J].上海交通大学学报(农业科学版),2008,26(3):233-236. |
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| 作者姓名： | 胡乔 赵凌侠 唐克轩 |
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| 作者单位： | 1. 上海交通大学,农业与生物学院,上海,200240;上海交通大学,生命科学技术学院,上海,200240
2. 上海交通大学,农业与生物学院,上海,200240 |
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| 基金项目： | 国家高技术研究发展计划(863计划)，上海市科委科技计划 |
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| 摘 要： | 将化学合成的人溶菌酶基因htYZ（444bp）构建到毕赤酵母分泌型表达载体pPICZ上；载体质粒用SacⅠ线性化后电击法转化毕赤酵母菌株GS115，在含Zeocin的抗性培养基筛选后PCR鉴定获得了9株重组菌株。重组菌株经甲醇诱导后取表达上清液进行SDS-PAGE电泳和Western blot分析。结果显示，重组hLYZ在毕赤酵母中成功表达，在诱导60h时表达量最高。用镍亲和层析柱纯化重组hLYZ，用Bradford法测得其含量约为324mg·L^-1。比浊法活性测定结果显示所表达的重组人溶菌酶活性达到4473U·mL^-1。本研究利用毕赤酵母分泌型表达载体成功表达了重组人溶菌酶，井探索了简单有效的纯化和活性测定方法，为利用毕赤酵母系统规模化生产重组人溶菌酶奠定了技术基础。
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| 关 键 词： | 人溶菌酶 毕赤酵母 生物活性 |
A Study on the Expression of Recombinant Human Lysozyme in Pichia pastoris |
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| HU Qiao,ZHAO Ling-xi,TANG Ke-xuan.A Study on the Expression of Recombinant Human Lysozyme in Pichia pastoris[J].Journal of Shanghai Jiaotong University (Agricultural Science),2008,26(3):233-236. |
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| Authors: | HU Qiao ZHAO Ling-xi TANG Ke-xuan |
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| Institution: | HU Qiao1,2,ZHAO Ling-xia1,TANG Ke-xuan1 (1.School of Agriculture , Biology,Shanghai Jiaotong University,Shanghai 200240,China,2.School of Life Science , Biotechnology,China) |
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| Abstract: | The hLYZ （human lysozyme） gene was chemically synthesized, then constructed into yeast secretion expression vector pPICZα. The constructed plasmid was linearized by Sac Ⅰ digestion and transformed into Pichia pastoris strain GS115 by electroporation method. Nine recombinant strains were successfully screened out by Zeocin and identified by PCR analysis. After induction by methanol, the supernatant from the induced recombinant P. pastoris strain was collected, then analyzed by SDS-PAGE and Western blot analyses. The results showed that the recombinant hLYZ protein was successfully expressed and the expression level reached the peak at 60 h after induction. The recombinant hLYZ was purified by Ni^＋ affinity chromatography and its yield was 324mg·L^-1 by Bradford assay. The bioactivity of the recombinant hLYZ was determined to be 4473 U ·mL^-l by turbidimetric method. The successful expression of the bioactive recombinant hLYZ using P. pastoris expression system and the＇exploration of the simple and efficient purification and bioactivity measurement method of the recombinant protein provide the basis for large-scale production of recombinant hLYZ using P. pastoris system. |
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| Keywords: | hLYZ Pichia pastoris bioactivity |
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