【摘要】 构建了人乙酰肝素酶基因系列真核表达载体 ,并在NIH3T3细胞中表达 ,以研究乙酰肝素酶的结构与功能的关系。采用PCR及GeneSOE的方法从人乙酰肝素酶cDNA中扩增得到hpaFL (Met1 Ile543 ) ,hpa10 9(Met1 Glu10 9)及hpasp5 0 (pro ,Met1 Ala3 5+Lys158 Ile543 ) ,然后插入真核表达载体 pcDNA 3.1中。采用脂质体介导法将其转染NIH3T3细胞 ,4 8h后RT PCR检测转染细胞中外源乙酰肝素酶基因的转录。结果 :酶切鉴定、测序结果以及RT PCR分析结果表明成功构建了重组表达载体 pcDNA hpaFL ,pcDNA hpa10 9及pcDNA hpasp5 0 ,并在NIH3T3细胞中有效转录。构建成功的乙酰肝素酶系列表达载体可为乙酰肝素酶结构与功能关系的研究奠定基础。更多还原

【Abstract】 To construct a series of human heparanase gene containing eukaryotic expressing vector and express them in NIH3T3 cell, hpaFL(Met 1-Ile 543),hpa109(Met 1-Glu 109) and hpasp50(pro,Met 1-Ala 35+Lys 158-Ile 543) were amplied by PCR and Gene SOE(Gene splicing by overlap extension).The fragment were cloned into pcDNA3.1 plasmids after digestion with restriction enzymes.Then NIH3T3 cells were transfected with the recombinant by liposome.The transcription and expression of heparanase in the transfected NIH3T3 cells were assayed by RT-PCR at 48 hours after transfection. A series of human heparanase gene containing eukaryotic expressing vectors were sucessfully constructed and those recombinants were transcripted in transfectants. The successful construction of pcDNA-hpaFL, pcDNA-hpa109 and pcDNA-hpasp50 could provide a rational basis for expression of the enzyme as well as research about the relationship between its structure and fuction.更多还原