在本实验中，来自酿酒酵母HS1185的胞外&#61538;－1，3－葡聚糖基因被插入TA克隆载体pMD-18中，并被转入大肠杆菌JM109中。重组质粒命名为pMDT-18-GLU。通过XhoI和NcoI双酶切质粒pMDT-18-GLU获得的&#61538;－1，3－葡聚糖基因片段插入pET22b(+)的XhoI和NcoI酶切位点。此重组质粒命名为pET22b/GLU。质粒pET22b-GLU被转入大肠杆菌BL21(DE3)，通过菌落PCR筛选出阳性克隆。 提取出来已经插入Beta－1，3－葡聚糖基因的pET22b-GLU质粒和pET22b(+)通过PCR扩增和限制性内切酶验证分析。PCR质粒...In this study the exo-&#61538;-1,3-glucanase gene (GenBank Accession No. X59259; EC No. 3.2.1.58) from Saccharomyces cerevisiae strain HS1185 was ligated with TA cloning vector pMDT-18 and transformed into Escherichia coli strain JM109. The constructed plasmid was named pMDT-18-GLU. Plasmid pMDT-18-GLU was digested with Xho I and Nco I restriction enzymes and the obtained fragment of &#61538;-1,3-...学位：工学硕士院系专业：化学化工学院化学工程与生物工程系_化学工程学号：2062007115407

BUYONDO JOHN PAUL

Xiamen University Institutional Repository

学校编码：10384 学号：20620071154071         硕  士  学  位  论  文         大肠杆菌表达重组Beta-1, 3-葡聚糖基因         Expression of Beta-1,3-Glucanase gene inEscherichia coli         BUYONDO JOHN PAUL         指导教师：卢英华            专业名称：化学工程               答辩日期：2009年7月   厦门大学博硕士论文摘要库厦门大学学位论文原创性声明                  本人呈交的学位论文是本人在导师指导下,独立完成的研究成果。本人在论文写作中参考其他个人或集体已经发表的研究成果，均在文中以适当方式明确标明，并符合法律规范和《厦门大学研究生学术活动规范(试行)》。                  另外，该学位论文为(                  )课题(组)的研究成果，获得(                    )课题(组)经费或实验室的资助，在(          )实验室完成。(请在以上括号内填写课题或课题组负责人或实验室名称，未有此项声明内容的，可以不作特别声明。)                           声明人(签名)：                            年   月   日厦门大学博硕士论文摘要库厦门大学学位论文著作权使用声明                  本人同意厦门大学根据《中华人民共和国学位条例暂行实施办法》等规定保留和使用此学位论文，并向主管部门或其指定机构送交学位论文(包括纸质版和电子版)，允许学位论文进入厦门大学图书馆及其数据库被查阅、借阅。本人同意厦门大学将学位论文加入全国博士、硕士学位论文共建单位数据库进行检索，将学位论文的标题和摘要汇编出版，采用影印、缩印或者其它方式合理复制学位论文。                  本学位论文属于：                  (     )1.经厦门大学保密委员会审查核定的保密学位论文，于年 月 日解密，解密后适用上述授权。                  (     )2.不保密，适用上述授权。                  (请在以上相应括号内打“√”或填上相应内容。保密学位论文应是已经厦门大学保密委员会审定过的学位论文，未经厦门大学保密委员会审定的学位论文均为公开学位论文。此声明栏不填写的，默认为公开学位论文，均适用上述授权。)                           声明人(签名)：                            年   月   日厦门大学博硕士论文摘要库摘  要                  在本实验中，来自酿酒酵母HS1185的胞外&#61538;－1， 3－葡聚糖基因被插入TA克隆载体pMD-18中，并被转入大肠杆菌JM109中。重组质粒命名为pMDT-18-GLU。通过Xho I和Nco I双酶切质粒pMDT-18-GLU获得的&#61538;－1， 3－葡聚糖基因片段插入pET22b(+)的Xho I和Nco I酶切位点。此重组质粒命名为pET22b/GLU。质粒pET22b-GLU被转入大肠杆菌BL21(DE3)，通过菌落PCR筛选出阳性克隆。提取出来已经插入Beta－1， 3－葡聚糖基因的pET22b-GLU质粒和pET22b(+)通过PCR扩增和限制性内切酶验证分析。PCR 质粒pET22b-GLU扩增出1337 bp的条带，与酿酒酵母中Beta－1， 3－葡聚糖基因大小一致。在酶切验证中，重组质粒pET22b/GLU被Xho I和Nco I双酶切，得到了5431 bp和1337 bp的两条带，分别为pET22b(+)和&#61538;－1， 3－葡聚糖基因。最后通过测序来进一步验证正确的基因序列被插入质粒中。测序结果显示，插入的基因序列与与酿酒酵母中&#61538;－1， 3－葡聚糖基因序列有98%的序列同源性。培养携带pET22b-GLU质粒的重组大肠杆菌BL21(DE3)，并通过IPTG诱导蛋白质表达。通过DNS测定残糖结果显示并没有酶活。但是酿酒酵母中&#61538;－1， 3－葡聚糖基因已经成功插入pET22b(+)质粒，并转入大肠杆菌BL21(DE3)中。         关键词：Beta－1, 3－葡聚糖基因；酿酒酵母；重组质粒厦门大学博硕士论文摘要库Abstract                  In this study the exo-&#61538;-1,3-glucanase gene (GenBank Accession No.X59259; EC No. 3.2.1.58) from Saccharomyces cerevisiae strain HS1185 wasligated with TA cloning vector pMDT-18 and transformed into Escherichia colistrain JM109. The constructed plasmid was named pMDT-18-GLU. PlasmidpMDT-18-GLU was digested with Xho I and Nco I restriction enzymes and theobtained fragment of &#61538;-1,3-glucanase gene was ligated with plasmidpET22b(+) at Xho I and Nco I restriction sites. The constructed plasmid wasnamed pET22b-GLU. Plasmid pET22b-GLU was transformed into Escherichiacoli strain BL21 (DE3) and the positive colonies were identified by performingcolony PCR.The extracted plasmid pET22b-GLU that contained the correct insert of the&#61538;-1,3-glucanase gene and plasmid pET22b(+) was confirmed using PCRamplification and restriction enzyme analysis. The performed PCR gave amplifiedfragments of about 1337 bp-the size which was in accordance with the originalsize of &#61538;-1,3-glucanase gene from Saccharomyces cerevisiae. For therestriction enzyme analysis, the recombinant plasmid pET22b-GLU was doubledigested with enzymes Xho I and Nco I and gave two bands of sizes 5431 bp and1337 bp for plasmid pET22b(+) and &#61538;-1,3-glucanase gene, respectively.Further confirmation of the correct insert was done by carrying out DNAsequencing of the cloned gene. The nucleotide sequence results showed highidentity of 98% to the original beta-1,3-glucanase gene from Saccharomycescerevisiae.The recombinant Escherichia coli strain BL21 (DE3) carrying plasmid pET22b-GLU was cultivated and induced with IPTG for protein expression. The resultsshowed that there was no activity using the DNS assay for reducing sugar.However, based on the results obtained, &#61538;-1,3-glucanase gene from厦门大学博硕士论文摘要库Saccharomyces cerevisiae strain HS185 was successfully cloned into plasmidpET22b(+) and transformed in Escherichia coli strain BL21(DE3).         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Expression of Beta-1,3-Glucanase gene in Escherichia coli

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Expression of Beta-1,3-Glucanase gene in Escherichia coli

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