NM23-pIRES2-EGFP质粒的构建与鉴定

发布时间：2018-11-13 16:03

【摘要】：目的:构建真核表达载体NM23-p IRES2-EGFP,并且分别做酶切与DNA测序鉴定准确性,为下一步探讨NM23基因对胃癌细胞的增殖凋亡与转移提供实验基础。方法:1.用TRIZOL法从人的正常胃粘膜组织中提取总的RNA,通过RT-PCR法扩增NM23基因,获得NM23基因的CDS序列;选择琼脂糖凝胶电泳鉴定,割胶纯化后回收NM23基因。2.与p MD18-T-simple克隆载体连接,得到p MD18-T-simple-NM23,送公司进行测序,测序结果与Genebank报道的人NM23基因序列一致。3.NM23基因与酶切处理后的载体pl RES2-EGFP连接,构建表达载体NM23-p IRES2-EGFP,用PCR、酶切与DNA测序检测重组质粒序列的准确性。结果:1.从人的正常胃粘膜组织中扩增出NM23基因,琼脂糖凝胶电泳显示RT-PC R产物在条带500-750bp之间,与文献的理论值534bp相符。2.与克隆载体p MD18-T-simple连接,测序鉴定与Genebank数据库记录的NM23基因的CDS区序列完全一致。3.使用克隆技术构建真核表达载体NM23-p IRES2-EGFP,并选用PCR、酶切与DNA测序进行验证,结果表明真核表达载体NM23-p IRES2-EGFP构建成功。结论:1.用TRIZOL法从人正常的胃粘膜组织中可扩增出大小为534bp的NM23基因,作为下一步构建载体的目的基因。2.NM23基因连接TA克隆载体,得到重组质粒p MD18-T-simple-NM23。3.成功构建质粒NM23-p IRES2-EGFP,经PCR、Xho I/Ba m HI双酶切、测序等证实了该重组质粒的准确性。
[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.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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