人PSMP重组蛋白在CHO细胞中表达、纯化及功能鉴定  

Expression,purification and functional identification of human PSMP recombinant protein in Chinese hamster ovary cells

作　　者：马靖[1] 裴晓磊 张扬[1] 王应[1] 

机构地区：[1]北京大学基础医学院免疫学系,北京大学人类疾病基因研究中心,卫生部医学免疫学重点实验室,北京100191

出　　处：《北京大学学报（医学版）》2014年第5期669-675,共7页Journal of Peking University:Health Sciences

基　　金：国家教育部博士点基金(20120001110001);国家“重大新药创制”科技重大专项(2011ZX09506-005);国家自然科学基金(31270915)资助~~

摘　　要：目的:构建新的具有趋化作用的人类细胞因子PSMP的真核表达载体,在仓鼠卵巢细胞(Chinese hamster ovary,CHO)中表达并纯化,为PSMP的功能机制研究奠定基础。方法:从pcDNA3.1-PSMP-myc/His中切下PSMPmyc/His片段,插入pMH3表达载体。利用电穿孔法将该表达载体转染CHO细胞,G418抗性筛选稳定克隆株。以Dot blot及Western blot法检测上清液中PSMP蛋白的表达,利用有限稀释法对抗性筛选出的混合克隆单克隆化,悬浮无血清大批培养工程细胞,通过镍亲和层析法纯化蛋白,SDS-PAGE电泳分析蛋白纯度,Boyden小室体外趋化实验分析蛋白功能活性。结果:PSMP-myc/His基因插入pMH3载体后成功构建真核表达载体pMH3-PSMP,转染CHO细胞后经2次克隆化获得稳定表达PSMP的基因工程细胞株。镍亲和层析纯化的重组蛋白PSMP纯度达95%以上且具有生物学活性。结论:成功构建了PSMP蛋白的真核表达载体,并获得其稳定表达的CHO细胞株,获得了较高纯度的、具有生物学活性的重组蛋白,为下一步PSMP功能和机制研究提供有用工具。Objective： To construct a new human chemotactic cytokine PSMP eukaryotic expression vector to express PSMP in Chinese hamster ovary（ CHO） cells and to obtain the purified recombinant PSMP protein for its functional mechanism study. Methods： PSMP-myc /His fragment, cut from pcDNA3. 1-PSMP-myc /His,was inserted into pMH3 expression vector. This expression vector was transfectedinto CHO cells by electroporation. Stable clone strains were selected by Geneticin resistance screening. The expressions of PSMP protein in the cell culture supernatant were measured by Dot blot and Western blot analysis. The monoclone was prepared from resistance screening polyclone by limiting dilution method. A large number of the engineering cells were cultured with serum-free medium and the protein in the cell culture supernatant was purified by nickel affinity chromatography. The purity of the PSMP protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis（ SDS-PAGE）.The functional activity of the protein was analyzed in vitro by Boyden chamber chemotaxis assay.Results： The eukaryotic expression vector pMH3-PSMP was successfully constructed by inserting PSMP-myc /His gene into pMH3 vector. After transfection of CHO cells,a stable expression of the PSMP gene engineering cell strain was obtained through twice cloning. The purity of the recombinant PSMP protein was 95% higher with bioactivity. Conclusion： The eukaryotic expression vector of PSMP protein is successfully constructed. The stable expression of PSMP is first obtained in CHO cell strain. The recombinantPSMP protein has higher purity and bioactivity,which provides a useful tool for further study of the functions and mechanisms of PSMP.

关 键 词：PC3-分泌微量蛋白 人 CHO细胞 遗传 重组 基因表达 分离和提纯 

分 类 号：R392.12[医药卫生—免疫学]