【作者】 吴建勇；

【导师】 孙祥明； 易小萍；

【作者基本信息】 华东理工大学 ， 生物化工， 2011， 硕士

【摘要】 本文首先将pFRT/lacZeo质粒转染CHO-K1细胞,经半乳糖苷酶活性检测筛选得到了含高活性位点的细胞株CHO/FRT2.通过定点突变对NESPl(专利报道序列)序列进行了密码子优化,得到了NESP2、NESP3,并构建了相关的表达载体pcDNA5/FRT/NESP。将表达载体与含FLP酶基因的pOG44质粒共转CHO/FRT2细胞,得到了高效表达重组NESP的细胞株CHO/FRT2/NESP:NESP1、NESP2、NESP3的表达水平分别为786 IU/ml、1315 IU/ml、1029 IU/ml,表明经过密码子优化的NESP2具有更高的蛋白表达水平。此外,将载体pcDNA5/FRT/NESP2转染各CHO/FRT细胞,ELISA检测结果显示NESP2的表达与半乳糖苷酶活性水平趋势相同,证实半乳糖苷酶作为筛选指示蛋白,可以用来筛选有效的蛋白表达热区。之后,通过蓝胶亲和层析,凝胶过滤,离子交换层析纯化了重组CHO/FRT2/NESP2细胞培养上清,得到了一定量初步纯化的重组NESP蛋白样品。通过SDS-PAGE电泳和Western blot分析证实,相对于原型EPO,NESP具有更高的分子量。等电聚焦分析结果也进一步证实NESP比EPO具有更高的唾液酸丰度,糖基化程度更高。更多还原

【Abstract】 The pFRT/lacZeo plasmid was transfected into CHO-K1 cells, and then obtained the CHO/FRT2 with high-level active domain by detecting the activity ofβ-galactosidase. NESP2 and NESP3 were obtained by codon optimization of NESP1 (the patent reported sequence) using site-directed mutation method, and then expression vector pcDNA5/FRT/NESP were constructed. The CHO/FRT2/NESP expressing high-level NESP was obtained by cotransfecting the expression vector pOG44 with flp gene into the CHO/FRT2. The expression-level of NESP1, NESP2 and NESP3 were 786 IU/ml,1315 IU/ml and 1029 IU/ml, respectively. The results indicated that codon optimization could improve the NESP expression.The expression vector pcDNA5/FRT/NESP2 was transfected into CHO/FRT cell lines, the results of ELISA demonstrated that expression of NESP2 and activity ofβ-galactosidase maintained the same trend, which indicated thatβ-galactosidase can be used as a good reporter to screen hot-spots of high-level protein expression.Then the cultured supernatant of CHO/FRT2/NESP2 was purified by blue-gel affinity chromatography, gel filtration chromatography (GFC) and ion exchange chromatography (IEC), SDS-PAGE and Western blot analysis indicated that compared with rHuEPO, NESP had a higher molecular weight. The result of isoelectrofocusing demonstrated that the most basic isoform of NESP with increased negative charge was more acidic than the most acidic isoform of rHuEPO.更多还原