摘要
将质粒ｐＢＸ－ＭＴ上的小鼠ＭＴ－ⅠｃＤＮＡ片段切下作为模板，通过ＰＣＲ方法删除该片段的非编码序列，将编码序列克隆到质粒ｐＢＳ－ＳＫ中，经ＤＮＡ序列测定后证明其克隆序列正确．再将ＭＴ－ⅠｃＤＮＡ编码序列插入到转移载体ｐＢａｃＰＡＫ８的ＢａｍＨⅠ和ＥｃｏＲⅠ位点之间，通过磷酸钙／ＤＮＡ共转染方法将其导入昆虫细胞Ｓｆ９中，以Ｗｅｓｔｅｒｎｂｌｏｔ和ＤｏｔＥＩＡ方法对表达产物进行了检测。
Mouse Metallothionein(MT) Ⅰ cDNA containing 149 bp non coding sequence and 186 bp coding sequence was cut from plasmid pBX MT and served as the PCR template.By using PCR mothod,the 5’ and 3’ flanking regions of non coding sequence were deleted,the coding sequence was cloned into plasmid pBS SK.Sequence analysis of Recombinant pBS MT confirmed that the cloned PCR product was the correct mouse MT Ⅰ cDNA.The mouse MT Ⅰ cDNA was inserted into transfer vector pBacPAK 8 and was transfected into Sf9 cells,a kind of insect cells,by DNA calcium phosphate cotransfected method.The level of expression of mouse MT Ⅰ cDNA was 1 mg/L detected by Western blot and Dot EIA.
基金
国家八六三生物高技术项目
