内切葡聚糖酶基因在巨大芽孢杆菌中重组表达载体的构建生物科学.docVIP

内切葡聚糖酶基因在巨大芽孢杆菌中重组表达载体的构建 生物科学 2003 级 廖俊华指导教师 陈 惠 教 授 摘要：以生物化学与分子生物学实验室克隆并测序的枯草芽孢杆菌C-36内切葡聚糖酶基因（Genbank 登录号：DQ782954）为模板，通过PCR将编码信号肽的序列去除，然后与pMD18-T Vector克隆载体连接，构建pMD18-T Vector亚克隆载体。对pMD18-T Vector质粒先进行BglⅡ单酶切，回收后再进行SphⅠ单酶切，通过两次单酶切胶回收内切葡聚糖酶基因（不含信号肽），并构建巨大芽孢杆菌重组表达载体pHEC-End，将重组表达载体转化到大肠杆菌DH5α中。通过对转化子进行菌落PCR和质粒的酶切检测证明成功构建了内切葡聚糖酶的巨大芽孢杆菌表达载体。 关键词：巨大芽孢杆菌，枯草芽孢杆菌， 内切葡聚糖酶 ，重组表达载体 Construction of eukaryotic Expression Vector of the gene of endoglucanase in Bacillus Megaterium LIAO Jun-hua Biological Science, Grade 2003 Directed by Chen Hui( Prof) Abstract: By the way of PCR, the sequence coming from endoglucanase gene (Genbank No. DQ782954 ) in B.subtilis C-36 strain and coding for the signal peptide of endoglucanase was removed.The endoglucanase gene was cloned and sequenced in the Laboratory of Biochemistry and Molecular Biology.Then the gene without the signal peptide sequence was ligated with the clone plasmid pMD18-T.The pMD18-T Vector was constructed. First, the pasmid was cuted by BglⅡ.Then,the pasmid was cuted by SphⅠ.By double enzyme-cut , the gene of endoglucanase in B.subtilis C-36 strain without signal peptide was recived. The eukaryotic expression vector pHEC-End was constructed.Eventually, the eukaryotic expression vector was transformed into competent E.coli DH5α . By the way of colony PCR and restriction enzyme digesting analysis, the eukaryotic expression vector of the gene of endoglucanase in Bacillus Megaterium was constructed succesfully. Keywords: Bacillus Megaterium，Bacillus subtilis，endoglucanase，eukaryotic expression vector 纤维素酶是一类能够降解纤维素，使之生成纤维二糖、葡萄糖等一系列酶的总称，按各酶功能的不同可以分为内切葡聚糖酶、外切葡聚糖酶和β-葡萄糖苷酶三大类[1]。纤维素被它降解后可转化为人类急需的能源、食物、化工原料，对于人类社会解决食物短缺和能源危机具有重大的现实意义[2]。随着中性纤维素酶和碱性纤维素酶在棉织品水洗整理工艺及洗涤剂工业中的成功应用，细菌纤维素酶制剂已显示出良好的使用性能和巨大的经济价值[3]。 纤维素酶成本高以及酶活力低已成为制约纤维素酶广泛应用的两大瓶颈，因此，构建高效表达的基因工程菌是降低纤维素酶成本、提高产量的有效手段。几乎所有已克隆到的纤维素酶基因都在大肠杆菌中得到了表达，但是都几乎存在着表达量低、分离提纯困难的缺陷。而芽孢杆菌因具有独特的蛋白分泌系统，使得在表达外源蛋白的宿主菌中深受青睐。其中枯草芽孢杆菌是应用较广泛的一种外源蛋白表达系统，并且已用于商业化生产酶制剂，例如，Geneco