發(fā)布時間：2018-11-13 16:03

【摘要】：目的:構(gòu)建真核表達載體NM23-p IRES2-EGFP,并且分別做酶切與DNA測序鑒定準確性,為下一步探討NM23基因?qū)ξ赴┘毎脑鲋车蛲雠c轉(zhuǎn)移提供實驗基礎(chǔ)。方法:1.用TRIZOL法從人的正常胃粘膜組織中提取總的RNA,通過RT-PCR法擴增NM23基因,獲得NM23基因的CDS序列;選擇瓊脂糖凝膠電泳鑒定,割膠純化后回收NM23基因。2.與p MD18-T-simple克隆載體連接,得到p MD18-T-simple-NM23,送公司進行測序,測序結(jié)果與Genebank報道的人NM23基因序列一致。3.NM23基因與酶切處理后的載體pl RES2-EGFP連接,構(gòu)建表達載體NM23-p IRES2-EGFP,用PCR、酶切與DNA測序檢測重組質(zhì)粒序列的準確性。結(jié)果:1.從人的正常胃粘膜組織中擴增出NM23基因,瓊脂糖凝膠電泳顯示RT-PC R產(chǎn)物在條帶500-750bp之間,與文獻的理論值534bp相符。2.與克隆載體p MD18-T-simple連接,測序鑒定與Genebank數(shù)據(jù)庫記錄的NM23基因的CDS區(qū)序列完全一致。3.使用克隆技術(shù)構(gòu)建真核表達載體NM23-p IRES2-EGFP,并選用PCR、酶切與DNA測序進行驗證,結(jié)果表明真核表達載體NM23-p IRES2-EGFP構(gòu)建成功。結(jié)論:1.用TRIZOL法從人正常的胃粘膜組織中可擴增出大小為534bp的NM23基因,作為下一步構(gòu)建載體的目的基因。2.NM23基因連接TA克隆載體,得到重組質(zhì)粒p MD18-T-simple-NM23。3.成功構(gòu)建質(zhì)粒NM23-p IRES2-EGFP,經(jīng)PCR、Xho I/Ba m HI雙酶切、測序等證實了該重組質(zhì)粒的準確性。
[Abstract]:Aim: to construct eukaryotic expression vector NM23-p IRES2-EGFP, and identify the accuracy of DNA sequencing and restriction endonuclease digestion, so as to provide the experimental basis for the further study of the proliferation, apoptosis and metastasis of gastric cancer cells by NM23 gene. Methods: 1. The total RNA, was extracted from human normal gastric mucosa by TRIZOL method and the CDS sequence of NM23 gene was amplified by RT-PCR method. The CDS sequence of NM23 gene was identified by agarose gel electrophoresis, and NM23 gene was recovered by gel electrophoresis. The p MD18-T-simple-NM23, gene was sequenced by ligating with the cloned vector of p MD18-T-simple. The result of sequencing was consistent with the sequence of human NM23 gene reported by Genebank. The 3.NM23 gene was ligated with the vector pl RES2-EGFP, which was digested by enzyme. The expression vector NM23-p IRES2-EGFP, was constructed to detect the accuracy of recombinant plasmid sequence by PCR, digestion and DNA sequencing. The result is 1: 1. NM23 gene was amplified from human normal gastric mucosa. Agarose gel electrophoresis showed that the RT-PC R product was between the bands of 500-750bp, which was consistent with the theoretical value of 534bp. 2. The sequence of the CDS region of the NM23 gene recorded in the Genebank database was completely consistent with that of the cloned vector p MD18-T-simple. 3. 3. The eukaryotic expression vector NM23-p IRES2-EGFP, was constructed by cloning technique, and PCR, was digested with DNA sequencing. The results showed that the eukaryotic expression vector NM23-p IRES2-EGFP was successfully constructed. Conclusion: 1. The NM23 gene with the size of 534bp can be amplified from normal human gastric mucosa by TRIZOL. The 2.NM23 gene is ligated into the TA clone vector as the next step to construct the vector, and the recombinant plasmid p MD18-T-simple-NM23.3. is obtained. The recombinant plasmid NM23-p IRES2-EGFP, was successfully digested by PCR,Xho I/Ba m HI and sequenced to confirm the accuracy of the recombinant plasmid.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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