Cloning of prealbumin cDNA and construction of the stable transfective cell line
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摘要: 建立真核表达重组体 pCDNA3 1(+) -PA的稳定转染细胞株。以人胚肝组织总RNA为模板通过RT-PCR获取全长PAcDNA。先将该序列克隆到 pGEM -TEasy载体 ,再亚克隆转入pCDNA3 1(+) ,限制性内切酶消化及DNA序列分析鉴定重组体构建正确后 ,用脂质体转染技术把它导入Hela细胞 ,经G4 18筛选建立稳定转染细胞株 ,真核表达重组体 pCDNA3 1(+) -PA构建成功 ,稳定转染细胞株中有PAcDNA表达。稳定转染细胞株的成功建立为进一步生产PA的基因工程产品奠定基础Abstract: To construct the stable transfective cell line with the eukaryotic expression vector for prealbumin cDNA.PA cDNA was obtained by RT-PCR of total RNA extracted from the human fetal liver.The sequence was inserted into pGEM-T Easy vector and then transfered into eukaryotic expression vector pCDNA 3 1 (+) .The recombination with forward insert were selected by restriction endonuclease digestion and confirmed correct by sequencing and then transfected into Hela cell line by using lipofectamine.The stable transfected cell lines were selected in medium containing geneticin (G418) .The full length prealbumin cDNA was inserted into the eukaryotic expression vector pCDNA3 1 (+) .The result of RT-PCR of total RNA exrtacted from the stable transfected cell lines showed that there exist the expression of PA cDNA in it.This work may pave the way for obtaining production of prealbumin in future.
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Key words:
- prealbumin /
- cDNA clone /
- transfect
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[1]ShujiM , ShuichiroM , KazunoriS .CloningandsequenceanalysisofcDNAforhumanprealbumin[J].JBiolChem, 1985, 263 (18) ∶8598-8603. [2]J·萨姆布鲁克等著金冬雁等译《分子克隆实验指南》[M].第二版北京∶科学出版社, 1992
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