Ⅰ：沙蚕纤溶活性蛋白编码基因的筛
发布时间：2018-08-02 19:50
【摘要】：①目的:构建沙蚕消化道上皮细胞的cDNA文库,并从中筛选、克隆和鉴定一种具有纤溶活性的蛋白编码基因序列。②方法:分离沙蚕消化道上皮细胞,使用Trizol试剂提取总RNA,纯化mRNA,用MMLV逆转录成cDNA,用Sfi I核酸内切酶酶切后连接到pDNR-Lib质粒中,构建cDNA文库。以SMART试剂盒中已设计好的引物行PCR扩增筛选获得多个克隆,将筛选成功的克隆进行核苷酸测序,将测得的序列所编码的相应蛋白使用分子生物学软件进行功能预测和二级、三级结构的模拟。③结果:获得沙蚕纤溶活性蛋白的编码序列以及插入此基因序列的重组菌。模拟结果显示该序列编码的蛋白与来源于哺乳动物的丝氨酸蛋白酶相似,具有该家族保守的催化位点。④结论:在原核表达系统中成功构建了插入此目的序列的重组菌,该基因序列编码的蛋白有可能成为一种新型的纤溶药物。
[Abstract]:Objective: to construct the cDNA library of digestive tract epithelial cells of silkworm, screen, clone and identify a protein coding gene sequence .2 with fibrinolytic activity: to isolate the epithelial cells of digestive tract of silkworm, silkworm. Total RNAs were extracted by Trizol reagent, mRNAs were purified, MMLV was reverse transcribed into cDNAs, then ligated into pDNR-Lib plasmids by Sfi I endonuclease. CDNA library was constructed. Several clones were obtained by PCR amplification with primers designed in the SMART kit. The cloned clones were sequenced. The corresponding proteins encoded by the detected sequences were predicted by molecular biology software. Results: the encoding sequence of fibrinolytic active protein and the recombinant bacteria inserted into the protein sequence were obtained. The simulation results show that the protein encoded by this sequence is similar to the serine protease from mammals and has the conserved catalytic site of this family. Conclusion: the recombinant bacteria inserted into the target sequence was successfully constructed in the prokaryotic expression system. The protein encoded by the gene sequence may become a new type of fibrinolytic drug.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
[Abstract]:Objective: to construct the cDNA library of digestive tract epithelial cells of silkworm, screen, clone and identify a protein coding gene sequence .2 with fibrinolytic activity: to isolate the epithelial cells of digestive tract of silkworm, silkworm. Total RNAs were extracted by Trizol reagent, mRNAs were purified, MMLV was reverse transcribed into cDNAs, then ligated into pDNR-Lib plasmids by Sfi I endonuclease. CDNA library was constructed. Several clones were obtained by PCR amplification with primers designed in the SMART kit. The cloned clones were sequenced. The corresponding proteins encoded by the detected sequences were predicted by molecular biology software. Results: the encoding sequence of fibrinolytic active protein and the recombinant bacteria inserted into the protein sequence were obtained. The simulation results show that the protein encoded by this sequence is similar to the serine protease from mammals and has the conserved catalytic site of this family. Conclusion: the recombinant bacteria inserted into the target sequence was successfully constructed in the prokaryotic expression system. The protein encoded by the gene sequence may become a new type of fibrinolytic drug.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
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