【作者】 黄琰；

【导师】 陈冠军；

【作者基本信息】 山东大学 ， 微生物学， 2005， 硕士

【摘要】 瑞氏木霉（Trichoderma reesei）纤维二糖水解酶Ⅰ（cellobiohydrolase Ⅰ）是目前研究的最为广泛的一种外切葡聚糖酶。其研究主要集中在两个方面,一方面是进一步阐释CBHI与结晶纤维素的作用机制,另一方面是对酶性质、特点、稳定性和催化活性等进行改造以满足生物工程应用的需要。CBHI在噬菌体展示系统中的展示,为进一步研究其作用机制和CBHI的定向进化提供了一个良好的研究平台。 本论文使用基因重组技术,分别构建了重组噬菌体表达质粒pCANTAB 5E—cbh1及pCANTAB 5E—cd,在噬菌体表面对纤维素外切酶CBHI全酶及其催化结构域（CD）进行了表达展示。 根据目的基因cbh1的cDNA序列设计两对引物,分别扩增cbh1全序列及其催化结构域序列cd,使之上游带SfiⅠ酶切位点,下游带NotⅠ酶切位点,以质粒pUCmT-cbh1为模板,进行PCR扩增反应,得到了长度分别为1.55kb和1.3kb的目的片段;将所得的DNA片段经NotⅠ和SfiⅠ双酶切后分别与噬菌粒载体pCANTAB 5E进行连接,然后导入大肠杆菌TGI中,用Amp^r筛选获得了阳性克隆,经双酶切鉴定,验证了构建的重组噬菌粒为pCANTAB 5E-cbh1和pCANTAB5E-cd。 用帮助噬菌体M13K07侵染验证后的TGI转化子（pCANTAB 5E-cbh1）,制备带有外源基因cbh1的重组噬菌体,酶联免疫吸附剂测定（ELISA）表明重组噬菌体对滤纸有明显的特异性吸附,说明展示后的CBHI的吸附特性没有改变。以pNPC为底物测定重组噬菌体的外切酶酶活,发现在重组噬菌体上表达的CBHI可水解pNPC底物,酶活为2.625×10^-14IU/CBHI分子,证明展示后的CBHI催化特性没有改变。将带有外源基因cbh1的重组噬菌体侵染大肠杆菌HB2151,通过IPTG诱导表达出可溶性的外源蛋白CBHI。SDS聚丙烯酰胺凝胶电泳结果显示,HB2151转导子与原始HB2151菌株所表达的蛋白在分子量为54kDa处出现明更多还原

【Abstract】 Cellobiohydrolase I from Trichoderma reesei is a most widely studied kind of exoglucanase. Studies about CBHI mostly focus on two aspects: one is the further explanation for the mechanisms of CBHI hydrolyzing crystalline cellulose; the other is to improve the properties, specificity, stability or catalytic activity of CBHI to make it suitable for biotechnological applications. The phage display of CBHI lays the foundation for these researches.This thesis constructed two recombinant phagemids pCANTAB 5E-cbhl and pCANTAB 5E-cd. Then transformed them into E.coli TGI and expressed CBHI on the phage tip by the infection of M13K07 helper phage.The cbh1 and cd (the DNA sequence of CBHI and the catalytic domain of CBHI) DNA fragments with Sfi I and Not I at 5’and 3’termini were prepared by PCR using plasmid pUCmT-cbh1 as template and two pairs of designed primers both carried Sfi I in upstream and Not I in downstream. After digested by both Not I and Sfi I, cbhl and cd DNA fragments were linked with phagemid PCANTAB 5E. The ligation products were transformed into competent TGI. Positive clones were screened. The recombinant phagemids pCANTAB 5E-cbhl and pCANTAB 5E-cd isolated andpurified from positive clones were confirmed by digesting with both Not I and Sfi I. The results showed that cbhl and cd DNA were linked with phagemid pCANTAB 5E successfully.The helper phage M13K07 was used to rescue the phagemid with cbhl gene insert from the transformed E.coli TGI cells. Enzyme linked immunosorbent assay (ELISA) showed that the recombinant phage specifically bound to filter paper; the exoglucanase activity of the displayed CBHI estimated with pNPC as the substrate was 2.625x 10" 14IU/CBHI molecule. The recombinant phage was also used to infect E.coli HB2151 cells for large-scale production of soluble recombinant CBHI. The molecular weight of the soluble CBHI protein was measured to be 54kDa, which was the same with the wild type CBHI from Trichoderma reesei. All these results showed that Cellobiohydrolase I was displayed by the Ml3 phage successfully.更多还原