促红细胞生成素基因转导骨骼肌细胞治疗肾性贫血的调控研究.pdfVIP

cDNA CHO RT-PCR (hEPO) pTRE2hyg pTRE2hyg-EPO pTRE2hyg-EPO Tet-On SD RT-PCRWestern hEPO EPO 1pTRE2hyg-EPO EPO 2EPO pTRE2hyg-EPO EPO : Objective To construct a tetracycline response eukaryotic expressing plasmid encoding recombination human erythropoietin gene (rhEPO); to study the rhEPO gene expression under the tetracycline gene expression system’ s regulating and controlling. Methods Amplified the rhEPO gene by RT-PCR from CHO cells which contain the total length cDNA of rhEPO genePCR products were cloned into eukaryotic expressing plasmid pTRE2hyg ,sequenced and done restriction endonuclease disgestion analysis. Transfer pTRE2hyg-EPO and Tet-On plasmid into rat skeletal muscle cell, measure rhEPO gene expression and biological activation of EPO by RT-PCR, Western blot and semisolid colony culture experiment, and observe the tetracycline gene expression system’s regulating and control functing to rhEPO gene expression. Results (1) pTRE2hyg-EPO eukarytic recombinant has been constructed successfully, and keep the same sequences with source rhEPO gene. (2)rhEPO can express in rat skeletal muscle cell, adjust and control under the tetracycline gene expression system. Producted erythropoietin have biological activation, it can obviously stimulates bone marrow cells growth. Conclusion the tetracycline gene expression system can adjust and control the pTRE2hyg-EPO gene expression at cell level. Produced erythropoietin have biological activation , and meet th