【摘要】 目的:构建hHGF-pCDNA3.1表达载体,在中国仓鼠卵巢细胞中获得具有生物学活性的重组人肝细胞生长因子蛋白的高效、稳定表达。方法:RT-PCR扩增人肝脏中hHGF全长cDNA片段,克隆入pCDNA3.1(+)真核表达载体中,构建hHGF-pCD-NA3.1重组质粒并转染中国仓鼠卵巢细胞,经筛选得到了高效、稳定表达hHGF的细胞克隆,采用RT-PCR和ELISA方法检测hHGF在中国仓鼠卵巢细胞中的表达。结果:酶切及测序结果表明重组质粒构建正确,RT-PCR显示细胞的rhHGF mRNA呈现高水平,ELISA检测hHGF在细胞中的分泌性表达,浓度达10ug/L。结论:成功地在中国仓鼠卵巢细胞中获得了hHGF蛋白的高效、稳定表达。为下一步将表达hHGF的细胞微囊化制备基因工程细胞,并移植用于相关疾病的基因治疗奠定了基础。更多还原

【Abstract】 Objective: To construct a plasmid carrying human hepatocyte growth factor(hHGF) gene and transfect it into Chinese hamster ovary(CHO) cells to establish a stable CHO system expressing high-lever rh HGF.Metheds: The full length cDNA of hHGF amplified from human liver mRNA by RT-PCR,was cloned into pCDNA3.1(+)vector to construct hHGF-pCDNA3.1 recombinant plasmid.The vector was then transfected into CHO cells with help of lipofectin liposome.The positive cell clone expressing hHGF was selected under the pressure of G418.Qualitative and quantitive detection of hHGF expression was performed through RT-PCR,immunofluorescence and ELISA.Results: The recombinant plasmid hHGF-pCDNA3.1 was identified by restriction endonuclease digestion and nucleotide sequencing.The expression of hHGF was verified and the concentration beyond 10μg/L.Conclusion: Base on the study,CHO system which expressing stable and highlever hHGF was established successfully,which lay the basis for microencapsulating CHO to form genetically engineered cell and for further clinical application.更多还原