豹蛙抗瘤酶与人血清白蛋白融合蛋白的毕赤酵母高效表达与活性测定  

Efficient Expression and Biological Activity Detection of Fusion Protein of Onconase with Human Serum Albumin in Pichia pastoris

作　　者：杨刚刚[1,2] 马诚凯 史世会 张全义 王泽 吕中原 王绪洋 许晓亚[1,2] 崔晴晴[1,2] 张继红[1,2] 丁一[1,2] 徐存拴 

机构地区：[1]河南师范大学生命科学学院,新乡453007 [2]河南省一科技部共建细胞分化国家重点实验室培育基地和河南省生物工程重点实验室,新乡453007 [3]河南新乡华星药厂,新乡453007

出　　处：《生物技术通报》2015年第10期222-229,共8页Biotechnology Bulletin

基　　金：国家"973"前期研究专项(2012CB722304);河南省自然科学基金项目(142300413212;132300413208);河南省重大科技攻关项目(111100910600)

摘　　要：豹蛙抗瘤酶(Onconase,ONC),又称豹蛙酶,对多种肿瘤细胞和实体瘤具有显著杀伤作用,为得到高表达的ONC,利用HSA在毕赤酵母系统中的表达优势,将HSA和ONC融合进行毕赤酵母表达并分离纯化。设计融合基因HSA-(Gly4Ser1)3-ONC,简称HSA-ONC,构建至3种表达载体p PIC9、p PIC9K、p PICZα-A并分别转染X-33、GS115和SMD1168 3种宿主菌,在摇瓶和10L规模优化表达条件和双水相偶联柱层析分离纯化目的蛋白并测定其生物学活性。结果显示,在1 L摇瓶规模下,p PICZα-A/X-33/HSA-ONC组合的表达量优于其他组合,且在p H7,温度23℃条件下诱导10 d,表达量达到最高(235 mg/L)。在10 L规模进行不同培养基条件的发酵,r HSA-ONC于p H 7的低盐基础盐培养基(Low salt basic salt mediu m,LS-BSM),甲醇浓度0.25%条件下诱导10 d,表达量达到最高(2.02 g/L)。经双水相萃取偶联DEAE离子交换层析可得到纯度≥95%、收率高于70%的r HSA-ONC。生物活性检测发现,r HSA-ONC在体外能抑制多种癌细胞增殖。r HSA-ONC的表达量较r ONC提高1倍(同等摩尔量情况下),同时在体外抑制多种癌细胞增殖。Onconase（ONC, also known as ranpirnase or P-30 protein）has weak RNase activity and significant toxic effect on various tumor cells and solid tumors. In order to obtain ONC owing highly efficient expression, we utilized the advantage of human serum albumin（HSA）expression in Pichia pastoris, had a fusion of HSA and ONC which was expressed in P. pastoris, then separated and purified the expressed protein. We designed the fusion gene according to the amino acid sequence of HSA-ONC and synthesized it based on the codons of P. pastoris, HSA-ONC gene was inserted into vector of p PIC9, p PIC9 K and p PICZα-A to form the recombinant plasmid p PIC9/HSA-ONC, p PIC9K/HSA-ONCand p PICZα-A/HSA-ONC, then the linearized recombinant plasmids were transformed into P. pastoris X-33, GSS115 and SMD1168 respectively. The expression conditions were optimized in the shake flask and 10-L bioreactor. The r HSA-ONC was purified by aqueous two-phase extraction coupling DEAE anion exchange chromatography, and the biological activity was detected by SRB assay. The highest r HSA-ONC production reached 235 mg/L in p PICZα-A/X-33/HSA-ONC combination under the condition of p H7 and 23℃in 1-L flask after induced 10 d, better than other combinations. In the 10-L bioreactor, when the induction was performed in the low basic salt medium of p H 7, and induced 10 d under 0.25% ethanol, the highest r HSA-ONC production could reach 2.02 g/L. The r HSA-ONC was purified by aqueous two-phase extraction coupling DEAE anion exchange chromatography, the purity was ≥ 95% and the yield was 70%. The results of detecting biological activity showed that the r HSA-ONC inhibited the proliferation of various tumor cells in vitro. Conclusively, the expression level of r HSA-ONC was twice as that of r ONC at the same molar dose, and r HSA-ONC inhibited the proliferation of various tumor cells in vitro.

关 键 词：豹蛙抗瘤酶 人血清蛋白 毕赤酵母高效表达 双水相萃取 癌细胞活性 

分 类 号：Q78[生物学—分子生物学]