本文選題：ALDH_2 + pUC57　； 參考：《昆明醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的：構(gòu)建pEGFP-N1-ALDH2真核表達載體及突變載體pEGFP-N1-ALDH2 (AAA),并轉(zhuǎn)染PC12細胞。驗證己轉(zhuǎn)染重組質(zhì)粒pEGFP-N1-ALDH2的PC12細胞高表達ALDH2,而突變重組質(zhì)粒則抑制ALDH2在PC12細胞中的表達。為我們今后的實驗“ALDH2對PC12細胞缺氧/復(fù)氧的保護作用”打下基礎(chǔ)。 方法：1、將現(xiàn)成購買的大鼠ALDH2基因進行PCR擴增后克隆到質(zhì)粒pUC57,構(gòu)建pUC57-Simple-ALDH2重組質(zhì)粒并轉(zhuǎn)入感受態(tài)細胞進行大量復(fù)制,提取陽性克隆,酶切鑒定并測序.2、將pUC57-Simple-ALDH2基因克隆到真核表達載體pEGFP-N1,構(gòu)建pEGFP-N1-ALDH2真核表達載體,酶切鑒定并測序。3、采用一步法點突變技術(shù)將ALDH2基因上的第506個氨基酸Glu(GAA)突變?yōu)長ys (AAA).4、用脂質(zhì)法將pEGFP-N1-ALDH2真核表達載體及突變質(zhì)粒轉(zhuǎn)染PC12細胞,經(jīng)G418篩選穩(wěn)定表達ALDH2及突變的細胞系 結(jié)果：(1)、成功地PCR擴增得到ALDH2基因片段；(2)、成功地構(gòu)建出pUC57-Simple-ALDH2重組質(zhì)粒,酶切鑒定測序正確。(3)、成功地構(gòu)建出pEGFP-N1-ALDH2真核表達載體,酶切鑒定測序正確。(4)、成功地構(gòu)建出pEGFP-N1-ALDH2(AAA)真核表達載體,PCR鑒定和測序正確。(5)、兩個重組質(zhì)粒成功轉(zhuǎn)染到PC12細胞,篩選出穩(wěn)定細胞系,經(jīng)過鑒定后pEGF-N1-ALDH2細胞系正常表達ALDH2蛋白,突變質(zhì)粒無表達。 結(jié)論：(1)、成功地將大鼠ALDH2基因進行PCR擴增,電泳結(jié)果顯示得到了正確擴增的DNA條帶。(2)、將正確合成的ALDH2基因克隆到質(zhì)粒pUC57上,測序沒有錯配的堿基對后,再將pUC57-ALDH2克隆到真核表達載體pEGFP-N1上,構(gòu)建了真核表達載體pEGFP-N1-ALDH2,酶切電泳結(jié)果及DAN測序均正確.(3)、成功地將ALDH2編碼區(qū)上的第506氨基酸殘基Glu(GAA)突變?yōu)長ys(AAA)得到突變型ALDH2基因,并成功構(gòu)建了突變型質(zhì)粒pEGFP-N1-ALDH2(AAA). (4)、兩個重組質(zhì)粒成功轉(zhuǎn)染到PC12細胞,篩選出穩(wěn)定細胞系,經(jīng)過鑒定后pEGF-N1-ALDH2細胞系正常表達ALDH2蛋白,突變質(zhì)粒無表達。
[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.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

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