小鼠MME基因真核表达载体的构建与鉴定

Construction and verification of mammalian cell expression vector of murine macrophage metalloelastase gene

目的 克隆小鼠巨噬细胞金属弹力酶 (MME)基因结构域Ⅰ和Ⅱ ,构建真核细胞表达载体 ,用于动物肿瘤原位种植模型的研究。方法 通过PCR选择性扩增MME基因结构域Ⅰ和Ⅱ ,克隆入 pGEM T载体中 ,限制性内切酶、DNA序列分析鉴定目的基因后 ,定向亚克隆到真核细胞表达载体pcDNA3 1中 ,并进行双酶切及PCR鉴定。 结果 以MMEcDNA为模板进行PCR扩增的特异性片段在 75 0bp和10 0 0bp之间。以此构建的pGEM T MME克隆载体和表达载体 ,经限制性内切酶酶切证实载体中带有MME目的基因片段 ;与小鼠MMEcDNA序列的碱基符合率为 99 6 3% ;证明MME基因已正确克隆到了真核细胞表达载体pcDNA3 1中。结论 成功构建了 pcDNA3 1 MME重组质粒 ,奠定了转基因研究MME表达与肿瘤生长转移关系的实验基础。

Objective To clone 819 bp cDNA fragment coding for domains Ⅰ and Ⅱ of MME and construct a eukaryotic expression vector. Methods A 840 bp cDNA fragment was amplified by polymerase chain reaction (PCR) method from pUC9-MME cDNA plasmid. The fragment was inserted into cloning vector pGEM-T and the recombinant pGEM-T-MME was identified by double digestion with restriction enzymes BamH Ⅰ and Xba Ⅰ and sequencing. The MME cDNA fragment was then subcloned into pcDNA3 1(+) plasmid. Results The pGEM-T-MME plasmid was digested into two fragments by 1% agarose gel electrophoresis with BamHⅠand XbaⅠ. They were consistent with theoretic values 3 0 kb and 832 bp, indicating that PCR products have been cloned into pGEM-T vector. The sequence of the insert was 99 63% identical to that in GeneBank. The recombinant pcDNA3 1-MME plasmid was separated into two bands(about 5 4 kb and 832 bp, respectively) in a 1% agarose gel using BamH Ⅰ and Xba Ⅰ, suggesting that MME gene fragment has been cloned into pcDNA3 1 vector correctly. Conclusion The recombinant mammalian cell expression vector pcDNA3 1-MME is successfully constructed. Our results lay a foundation for further transgenic animal model study on the relationship between expression of MME and growth and metastasis of colon cancer.