摘要
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.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1999年第3期378-382,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家九五科技攻关项目
国家教委博士点基金