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目的:为了探讨体内分泌表达胰高血糖素样肽1(GLP-1)治疗2型糖尿病的可行性,构建可分泌表达胰高血糖素样肽1的cDNA克隆。方法:实验于2005-09/2006-04在西安华广生物工程公司实验室进行。采用非对称互补引物/膜板法,体外合成两端含有酶切位点的胰高血糖素样肽1的cDNA片段,通过用NaeI和XhoI内切酶消化从NT4cDNA克隆中获取NT4的信号肽和前导序列,通过T4连接酶连接构建分泌表达胰高血糖素样肽1的cDNA克隆。该克隆的特点是确保信号肽酶识别点的正确性。结果:测序证实克隆的胰高血糖素样肽1的cDNA与GeneBank提供的序列完全一致;经限制性内切酶物理图谱方法证实已经成功地将胰高血糖素样肽1cDNA重组到含NT4的信号肽和前导序列下游。DNAsis软件分析结果表明该克隆编码具有信号肽酶识别点的NT4-GLP-1融合蛋白。结论:成功构建了能够分泌表达胰高血糖素样肽1的融合蛋白的cDNA克隆。
OBJECTIVE: To investigate the feasibility of in vivo secretion of glucagon-like peptide 1 (GLP-1) for type 2 diabetes mellitus, a cDNA clone secreting glucagon-like peptide 1 was constructed. Methods: The experiment was performed in Xi’an Hua Guang Bioengineering Laboratory from September 2005 to April 2006. CDNA fragments of glucagon-like peptide 1 containing restriction enzyme sites at both ends were synthesized in vitro by asymmetric complementary primer / membrane method. The signal peptide of NT4 was obtained from NT4 cDNA clones by digestion with NaeI and XhoI endonucleases and Leader sequence, a cDNA clone secreting and expressing glucagon-like peptide 1 was constructed by T4 ligase. The cloning is characterized by ensuring the correctness of signal peptidase recognition points. Results: Sequencing confirmed that the cloned glucagon-like peptide 1 cDNA was identical to that provided by GeneBank; restriction endonuclease physical mapping confirmed that the glucagon-like peptide 1 cDNA was successfully recombined to NT4 containing signal Peptides and leader sequences downstream. DNAsis software analysis indicated that the clone encodes the NT4-GLP-1 fusion protein with a signal peptidase recognition site. CONCLUSION: cDNA clones capable of secreting a fusion protein that expresses glucagon-like peptide 1 were successfully constructed.