用加端PCR技术改造h－IGF－1cDNA

Amplification of hIGF-1 Coding Sequence by PCR Technique

用ＤＮＡ合成仪合成了分别带有ＰｓｔＩ位点和ＳａｌＩ位点及终止密码子的２个用于扩增ｈＩＧＦ－１ｃＤＮＡ的ＰＣＲ引物．利用合成的引物，７００ｂｐ长的ｈＩＧＦ－１ｃＤＮＡ模板和Ｔａｑ聚合酶进行ＰＣＲ扩增．扩增产物经电泳鉴定后克隆进Ｍ１３ｍｐ１８载体，进行核苷酸序列分析．结果显示：ＰＣＲ产物含已发表的ｈＩＧＦ－１成熟蛋白的编码序列和５＇端的ＰＳｔＩ位点及３＇端的ＳａｉＩ位点及终止密码ＴＡＧ．用加端ＰＣＲ技术成功地扩增和改造了ｈＩＧＦ－１的编码序列．

Two primers containing the Pst I site and Sal I site and stop codon respectively were synthesized and used to amplify a 232 hp DNA sequence coding for mature hIGF-1 protein with the 700 bp hIGF-1 cDNA fragment as template by Taq polymerase.The amplified DNA fragment was cloned into M13mp18 vector and sequenced by Sanger-s dideoxy chain termination method. The sequencing data show that the nucleotide sequence of the cloned fragment is the same as hIGF-1 coding sequence published and that we have successfully remoulded the hIGF-1 protein coding region with Pst I site at 5' end and Sal I site and stop codon at 3' end using the add-on PCR technique.