【作者】 吴穗洁；

【导师】 章晓波；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 陆地资源开发已日趋饱和的今天,海洋资源的开发和利用日渐成为国际上关注的焦点。嗜热微生物作为其中重要的组成部分,不仅在生物系统进化中具有特殊地位,蕴涵生命进化历程中丰富的信息;其代谢过程中特殊的产物,尤其是嗜热酶在工业上有着广阔的应用前景,具有重要的研究意义。同时,对高温噬菌体的深入研究,将有助于了解其独特的生物化学和分子生物学特征,特别是相关结构基因及寻找强启动子方面的研究,将为嗜热菌表达系统的人工载体构建奠定基础。本研究从太平洋深海热液区筛选分离到一株高产木聚糖酶的嗜热菌MT-1,根据细菌16S rDNA序列分析,该嗜热菌属于Geobacillus sp.。从中克隆到一个编码331个氨基酸的木聚糖酶基因,并在Escherichia coli中高效表达。酶学性质测定结果表明,重组木聚糖酶的性质与粗酶类似,其最适温度和最适pH值分别为70℃和7.0,当温度高达90℃时,仍具有90%以上的酶活,且在pH 5.5-10范围内均可检测到较强的酶活。除了SDS对重组木聚糖酶酶活具有显著的抑制作用外,其他酶抑制剂PMSF、EDTA、2-ME、DTT（1mM）和去污剂Tween 20、Chaps、Triton X-100（0.1%）对酶活均无显著影响。金属离子对重组木聚糖酶酶活的影响研究表明,一价金属离子（Li⁺、Na⁺、K⁺）对酶活略有促进作用,除Mg²⁺、Ca²⁺、Ba²⁺以外的二价和三价金属离子普遍对酶活有强烈的抑制作用。目前,没有适合于嗜热细菌进行蛋白重组表达的载体,为了探索采用噬菌体构建嗜热菌表达系统,在本实验室已获得的深海嗜热细菌噬菌体GSVE1的基础上,研究了噬菌体尾部蛋白的功能。根据噬菌体GSVE1全基因组测序结果和开放阅读框的分析结果,对其中预测编码噬菌体结构蛋白的vp192基因开展研究,以噬菌体基因组为模板,克隆了vp192基因,并在E.coli中进行VP192蛋白的表达和纯化,制备抗体;Northern blot和Western blot结果显示,在噬菌体感染宿主菌后不同时期,vp192基因的转录和表达水平略有差异,大约在4h时达到最大值;免疫电镜结果显示,vp192基因如预测,编码噬菌体尾部的一个重要结构蛋白。进一步分析表明,该vp192基因处于一个尾部相关基因高度有序排列的功能基因簇的起始端,其下游的一段序列可能通过移码翻译的机制编码两个具有相同N末端的蛋白,随之对该移码翻译机制进行初步探索,将该段DNA序列克隆到带有GST标签的pGEX-4T-2表达载体中,并用GST单克隆抗体进行检测,发现同时存在两种蛋白,分子量大小与预期一致,分别为38 kDa和49 kDa,且两种蛋白在表达量上存在一定比例。更多还原

【Abstract】 The resources in the ocean are the most abundant on the earth, and become more and more attractive to people, since the terrestrial resources have been fully developed these years. Thermophiles are adapted to survive at high temperature. They are of great importance in the study of biological systematic evolution. Moreover, they can produce unique biocatalysts that function under extreme conditions comparable to those prevailing in various industrial processes. The enzymes from thermophiles, therefore, are of great interest for industrial applications. Studies on thermophilic bacteriophages suggest that bacteriophages are not only helpful for understanding the life, but also of great potential use in industry to serve as an artifical expression vector.During the isolation of thermophiles, a xylanase-producing thermophilic strain MT-1, which was assigned to Geobacillus sp. MT-1 based on 16S rDNA sequence, was isolated from a deep-sea hydrothermal field in east Pacific. Subsequently, a xylanase gene encoding a 331 amino-acid peptide from this isolate was cloned and expressed in Escherichia coli. The recombinant xylanase exhibited maximum activity at 70°C and had an optimum pH of 7.0. It was active up to 90°C and showed activity over a wide pH ranging from 5.5 to 10.0. The crude xylanase presented similar properties in temperature and pH to those of the recombinant xylanase. The recombinant xylanase was stable in 1 mM of enzyme inhibitors （PMSF, EDTA, 2-ME or DTT） and in 0.1% detergents （Tween 20, Chaps or Triton X-100）, whereas, it was strongly inhibited by sodium dodecyl sulfate （SDS） （1 mM）. In addition, its catalytic function was stable in the presence of Li⁺, Na⁺ or K⁺. However, it was strongly inhibited by Ni²⁺, Mn²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺ and Al³⁺ （1 or 0.1 mM）.Based on the complete genome sequence and ORF analysis of deep-sea thermophilic bacteriophage GSVE1, the ORF （termed as vp192 gene） that probably encoded a structural protein was identified. The gene encoding a 192 amino-acid peptide was cloned and expressed in E. coli as a fusion protein with glutathione S-transferase （GST）. Specific antibody was raised using the purified VP192 protein which was digested by thrombin. Temporal analysis showed that the vp192 gene was transcripted and expressed at various infection stages, and achieved the maximum amount at 4h. After incubation with anti-VP192 IgG followed by labelling with gold-labelled secondary antibody, the gold particles could be clearly found surrounding the tail of GSVE1 virions. This provided visual evidence that the vp192 gene encoded the phage tail protein. Further study was conducted to verify the translational frameshifting. The results revealed that the sequence downstream the vp192 gene encoded two proteins with the same N-terminal end. The ratio of the two proteins seemed to be a fixed number.更多还原