人血小板生成素重组载体的构建与体外表达

Construction and in vitro Expression of the Human Thrombopoietin Gene Containing Recombinant Plasmid

应用基因克隆技术，以人ＴＰＯｃＤＮＡ为目的基因，构建真核细胞表达重组体ＶＲＴＰＯ，藉脂质体将其转染于ＮＩＨ３Ｔ３细胞，应用ＰＣＲ、ＲＴ－ＰＣＲ及Ｗｅｓｔｅｒｎ印迹等技术对其转染及表达情况进行鉴定．结果表明：ＶＲＴＰＯ构建成功，在ＮＩＨ３Ｔ３细胞可表达人ＴＰＯ。

Direct injection of naked plasmid DNA encoding the gene of interest is a clinically practical gene transfer technique.A rhTPO cDNA containing plasmid vector was reconstructed,it was expected to have a higher expression efficacy in skeletal muscle in an attempt to explore the feasibility to use this technique in the TPO genetic therapeutics.By means of the routine recominant DNA techniques,the full length human TPO cDNA was cut from pUC118/TPO.With the targeting gene and through two subclonings,it was inserted into the final vector VR1012,so the needed eukaryotic expression vector,VRTPO,was generated.Following identification by restriction endonuclease cleavage and PCR,the recombinant vector was transiently transfected into NIH3T3 fibroblasts via lipofection.The transduction and expression of the vector in the cells was analyzed through PCR,RT PCR and Western blotting.The results demonstrated that the vector had successfully constructed,and the observations suggested that it had expression both at mRNA and protein level in NIH3T3 cells.The expressed rhTPO immigrated between the 48 3 kD and 82 0 kD of protein standard on SDS PAGE,and would be possibly glycosylated.All of these results suggested that it may be used for further in vestigation of in vivo TPO gene therapy on experimental animals.