【作者】 周培；

【导师】 邹竹荣；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 蜘蛛丝(spider silk)是一类天然蛋白质纤维,具有独特的机械性能(高强度、高弹性和高断裂功等)和卓著的生物学特性(生物可降解性和与生物组织的相容性等)。它在生物医学、材料、纺织和军事等领域都有着很大潜在的应用价值。但是由于蜘蛛无法驯养和天然蜘蛛丝产量少等原因,大量获取蜘蛛丝惟有通过基因工程手段,才能满足蜘蛛丝潜在的应用需求。本工作根据已报道的蜘蛛丝蛋白的氨基酸序列,结合蜘蛛丝蛋白的模块结构特性和功能的关系,优化设计了一种新型蜘蛛丝蛋白单元,它理论上集高强度、高弹性等蜘蛛丝主要特性于一体。编码该蛋白单元的基因SPS通过引物重叠延伸法得到了全长合成,为405 bp,并通过了测序验证。将SPS基因克隆到pET-32a(+)中,与其N端的硫氧还蛋白(TrxA)融合构建成表达载体pET-SPS1;进一步通过同尾酶法构建了蜘蛛丝蛋白基因SPS的多个串联体表达载体pET-SPS(2～8)。然后,所有的含重组蜘蛛丝蛋白基因的表达载体在大肠杆菌BL21(DE3)中进行了诱导表达,并且通过pET-32a(+)自身携带的His标签亲合层析纯化了融合表达的重组蜘蛛丝蛋白。结果表明,所有在大肠杆菌中融合表达的重组蜘蛛丝蛋白几乎全部可溶。其中,1～3串联体蜘蛛丝蛋白基因能够有效表达,但随着串联数的进一步增加,蜘蛛丝蛋白基因融合表达效率下降。另外,本工作进一步选用植物系统来表达重组蜘蛛丝蛋白。其中构建了含蜘蛛丝蛋白基因的植物表达载体pBI-SPS(5～7),通过冻融法将重组质粒转化到根癌农杆菌LBA 4404感受态细胞中。然后,通过烟草叶盘农杆菌浸染和抗性组织培养,得到了相应的转化再生植株。总之,本工作成功地获得按照意愿设计的一种新型蜘蛛丝蛋白基因,构建了它的大基因片段,并初步通过大肠杆菌和植物表达系统来表达,为未来这种新型蜘蛛丝的应用研究打下了基础。更多还原

【Abstract】 Spider silks,being a class of nature protein fibers,can be widely used in the biomedical,material,textile and military areas,because of their unique mechanical features of high strength,elasticity and high energy to break,and eminent biological peculiarities in terms of biodegradability and compatibility to the biological tissues.Because of inevitable problems in spider cultivation to produce natural spider silks, large amount of spider silks for their applications can only be obtained through genetic engineering.In this paper,an genetic spider silk protein unit was designed on the basis of amino acid sequences which is previously reported,in which the features of structural modules and their functional correlations were particularly concerned.Theoretically,this new-type spider silk protein is particularly endowed with two main pecaularities of spider silks which are high-intensity and high-elaticsity.The gene SPS(405 bp)encoding this silk protein unit was delicately synthesized by the manners of overlapped primer extension, finally verified by DNA sequencing.SPS gene was fused behind the tag of thioredoxin(TrxA)in plasmid pET-32a(+)to generate the expression vector pET-SPS1.This vector was further used as the backbone for creation of a set of expression vectors,pET-SPS(2～8)having different multimers of SPS gene by means of isocaudarners,all SPS vectors were expressed in E.coli strain, BL21(DE3)by IPTG induction,and the expression of fusion proteins of spider silk were purified by His-tag affinity chromatography.All SPS fusion proteins expressed in E.coli are almost fully soluble.The vectors with 1-3 multimers of SPS could be efficiently expressed,while more increased multimers could only cause worse expression.In addition,plant host was used in this work to produce recombinant spider silk proteins.For that,a few of SPS plant expression vectors of pBI-SPS(5～7)were generated,and transformed into Agrobacterium strain LBA 4404 by freeze-thaw method. These engineered strains were subsequently transformed by tobacco plants leaf disc infiltration.Finally,several corresponded lines of regenerated tobacco plants were obtained through tissue culturing with strict antibiotic selection.In one word,this work was completed with the success of gaining the gene encoding a new- type of artificial spider silk protein with distinct features of high intensity and elasticity.Prelimilarily expression of this gene and its mutimers were investigated by using the systems of E.coli and plants,which could Laying the foundation of applications of this new-type silk protein in the future.更多还原