| 摘要: |
| 【目的】建立一种对人抗凝血酶(AT)Cys248位点进行定点突变和真核表达的可靠方法，为突变体的结构和功能研究提供原始材料。【方法】以人AT cDNA序列为模板，设计1对反向扩增引物，进行反向PCR反应；PCR产物经连接、测序后，克隆至表达质粒pcDNA3.1(+)-GFP上，构建重组真核表达载体pcDNA3.1(+)-mAT-GFP；用重组载体介导突变基因在山羊乳腺上皮(GME)细胞内表达，收集细胞培养上清液进行表达产物的定性、定量检测。【结果】突变基因经测序证实与预期结果一致，且在GME细胞内得到高效表达，细胞培养液上清中的突变体质量浓度为（526±17.3） mg/L。【结论】反向PCR法是对-AT基因进行定点突变的可靠方法，AT-突变基因在GME细胞内可高效表达。 |
| 关键词: 反向PCR 人抗凝血酶 Cys248 定点突变 真核表达 |
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| 基金项目:湖南省自然科学基金 衡阳联合基金项目(13JJ8021)；衡阳市科技计划一般项目（2012KN24）；衡阳师范学院产学研用一般项目（12CXYY07）；衡阳师范学院大学生研究性学习和创新性实验计划项目（CXZX1307） |
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| Construction and eukaryotic expression of human antithrombin Cys248 site mutant |
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| Abstract: |
| 【Objective】This study aimed to explore a reliable method for site-mutation and eukaryotic expression of human antithrombin (AT) Cys248 mutant,and to provide the original material for the structural and functional research of AT mutant.【Method】AT cDNA sequence was taken as template and a pair of inverse amplification primers were designed for inverse polymerase chain reaction (PCR).The PCR products were connected,sequenced and cloned into the expression plasmid of pcDNA3.1(+)-GFP for the construction of recombinant eukaryotic expression vector pcDNA3.1(+)-mAT-GFP.Mutated gene was mediated by the recombinant plasmid and expressed in the goat mammary epithelial (GME) cells,and then the cell culture medium was collected and performed for qualitative and quantitative detection of the mutant.【Result】AT gene was successfully mutated by inverse PCR method,and it was expressed in GME cells efficiently.The concentration of the AT mutant was (526±17.3) mg/L in the cell culture medium.【Conclusion】Inverse PCR method was reliable for site directed mutation of AT gene,and successfully gaining of AT Cys248 mutants in GME cells medium provided the convenience for its structural and functional research in future. |
| Key words: inverse PCR human antithrombin Cys248 site directed mutation eukaryotic expression |
