真核表达载体pEGFP-N1-ALDH2及其突变质粒的构建和鉴定

发布时间：2018-05-17 16:26

本文选题：ALDH_2 + pUC57　；参考：《昆明医学院》2011年硕士论文

【摘要】：目的：构建pEGFP-N1-ALDH2真核表达载体及突变载体pEGFP-N1-ALDH2 (AAA),并转染PC12细胞。验证己转染重组质粒pEGFP-N1-ALDH2的PC12细胞高表达ALDH2,而突变重组质粒则抑制ALDH2在PC12细胞中的表达。为我们今后的实验“ALDH2对PC12细胞缺氧/复氧的保护作用”打下基础。方法：1、将现成购买的大鼠ALDH2基因进行PCR扩增后克隆到质粒pUC57,构建pUC57-Simple-ALDH2重组质粒并转入感受态细胞进行大量复制,提取阳性克隆,酶切鉴定并测序.2、将pUC57-Simple-ALDH2基因克隆到真核表达载体pEGFP-N1,构建pEGFP-N1-ALDH2真核表达载体,酶切鉴定并测序。3、采用一步法点突变技术将ALDH2基因上的第506个氨基酸Glu(GAA)突变为Lys (AAA).4、用脂质法将pEGFP-N1-ALDH2真核表达载体及突变质粒转染PC12细胞,经G418筛选稳定表达ALDH2及突变的细胞系结果：(1)、成功地PCR扩增得到ALDH2基因片段；(2)、成功地构建出pUC57-Simple-ALDH2重组质粒,酶切鉴定测序正确。(3)、成功地构建出pEGFP-N1-ALDH2真核表达载体,酶切鉴定测序正确。(4)、成功地构建出pEGFP-N1-ALDH2(AAA)真核表达载体,PCR鉴定和测序正确。(5)、两个重组质粒成功转染到PC12细胞,筛选出稳定细胞系,经过鉴定后pEGF-N1-ALDH2细胞系正常表达ALDH2蛋白,突变质粒无表达。结论：(1)、成功地将大鼠ALDH2基因进行PCR扩增,电泳结果显示得到了正确扩增的DNA条带。(2)、将正确合成的ALDH2基因克隆到质粒pUC57上,测序没有错配的碱基对后,再将pUC57-ALDH2克隆到真核表达载体pEGFP-N1上,构建了真核表达载体pEGFP-N1-ALDH2,酶切电泳结果及DAN测序均正确.(3)、成功地将ALDH2编码区上的第506氨基酸残基Glu(GAA)突变为Lys(AAA)得到突变型ALDH2基因,并成功构建了突变型质粒pEGFP-N1-ALDH2(AAA). (4)、两个重组质粒成功转染到PC12细胞,筛选出稳定细胞系,经过鉴定后pEGF-N1-ALDH2细胞系正常表达ALDH2蛋白,突变质粒无表达。
[Abstract]:Aim: to construct a pEGFP-N1-ALDH2 eukaryotic expression vector and a mutant vector pEGFP-N1-ALDH2 AAA, and to transfect PC12 cells. The results showed that PC12 cells transfected with recombinant plasmid pEGFP-N1-ALDH2 overexpressed ALDH2, while mutant recombinant plasmids inhibited the expression of ALDH2 in PC12 cells. To lay a foundation for our future experiment, "the protective effect of ALDH2 on hypoxia / reoxygenation of PC12 cells". Methods: 1. The rat ALDH2 gene was amplified by PCR and cloned into plasmid pUC57. the recombinant plasmid of pUC57-Simple-ALDH2 was constructed and transferred into the receptive cells for a large number of replication, and the positive clones were extracted. PUC57-Simple-ALDH2 gene was cloned into eukaryotic expression vector pEGFP-N1, and pEGFP-N1-ALDH2 eukaryotic expression vector was constructed. Restriction endonuclease digestion was used to identify and sequence .3. the 506th amino acid GluAGAA on ALDH2 gene was mutated into Lys AAA. 4. The eukaryotic expression vector and mutant plasmid of pEGFP-N1-ALDH2 were transfected into PC12 cells by lipid method. The stable expression of ALDH2 and mutant cell lines were screened by G418. Results the ALDH2 gene fragment was successfully amplified by PCR, and the recombinant plasmid of pUC57-Simple-ALDH2 was successfully constructed. The pEGFP-N1-ALDH2 eukaryotic expression vector was successfully constructed by restriction endonuclease digestion and sequencing. The eukaryotic expression vector pEGFP-N1-ALDH2AAA was successfully constructed and sequenced correctly. The two recombinant plasmids were successfully transfected into PC12 cells, and stable cell lines were screened out. After identification, the pEGF-N1-ALDH2 cell lines expressed ALDH2 protein normally. The mutant plasmid was not expressed. Conclusion the rat ALDH2 gene was successfully amplified by PCR. The result of electrophoresis showed that the correctly amplified DNA band was obtained. The correctly synthesized ALDH2 gene was cloned into the plasmid pUC57 and sequenced without mismatched base pair. PUC57-ALDH2 was cloned into eukaryotic expression vector pEGFP-N1, and the eukaryotic expression vector pEGFP-N1-ALDH2 was constructed. The results of restriction endonuclease electrophoresis and DAN sequencing were correct. GlugaA, the 506th amino acid residues in the ALDH2 coding region, was successfully mutated to Lys-AAA to obtain the mutant ALDH2 gene. The mutant plasmid pEGFP-N _ 1-ALDH _ 2 was constructed successfully. Two recombinant plasmids were successfully transfected into PC12 cells, and stable cell lines were screened. After identification, the pEGF-N1-ALDH2 cell lines expressed ALDH2 protein normally, but the mutant plasmids did not.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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