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

Abstract: 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 transfected into 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 recombinant PSMP protein has higher purity and bioactivity, which provides a useful tool for further study of the functions and mechanisms of PSMP.

Key words: PC3-secreted microprotein, human, CHO cells, Recombination, genetic, Gene expression, Isolation and purification