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Stable Expression of the Recombinant Human Membrane Form Klotho Protein in CHO Cells

Author: ZhongHui
Tutor: YanZuoQun
School: Jilin University
Course: Medical and Biological Engineering
Keywords: People klotho gene People Klotho protein Membrane type CHO cells Stably transfected
CLC: R346
Type: Master's thesis
Year: 2007
Downloads: 137
Quote: 0
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Abstract


In this experiment, the human genomic DNA as a template to construct cloning plasmid pEGFP-C1 - hmkl gene splicing : ① by PCR method from pcDNA3.1/zeo ()-hskl first three exons , the cloned and introduces a SalI site into the vector pEGFP-C1 . ( 2 ) using human genomic DNA as template using the PCR method to obtain hmkl gene EX4 vector pEGFP - C1 - hmkl EX ( 1 2 3 ) connected construct into pEGFP - a C1 - hmkl EX ( 1 2 3 4 ) , while introduction of EcoRI and AflII enzyme cleavage sites . ③ human genomic DNA as template by PCR method to get hmkl gene ex5 to the connection vector pEGFP -C1 - hmkl ex ( 1 2 3 4 ) eventually build into a cloning plasmid pEGFP - C1- hmkl . BamHI and XhoI enzyme Qieke Long plasmid pEGFP-C1-hmkl, to get complete hmkl gene construct eukaryotic the expression plasmid pcDNA3.1/zeo ( ) - hmkl . Extraction and purification kit Qiagen, endotoxin - free plasmid purified plasmid was confirmed by sequencing . Cationic polymer transfection reagent ( ) - hmkl into CHO cells expressing plasmid pcDNA3.1/zeo ; 600μg/ml zeocin - containing DMEM complete medium ( containing 10% fetal calf serum ) screened positive cell clones ; containing 300μg / ml zeocin DMEM complete culture medium to maintain the expansion of culture positive clone cells ; stably expressing hmKL protein in the cell line CHO - hmKL . The results show that in this experiment for the first time to obtain a stable cell strain CHO - hmKL expression the natural structure hmKL protein can be achieved , so that the recombinant expression of proteins having the natural structure hmKL . The success of the natural structure of recombinant hmKL protein obtained to explore KL exact biological function of the protein provides a prerequisite .

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