次壶腹腺丝蛋白功能模块的研究  

The Research of MiSp Modules

作　　者：贾小娜[1] 陈格飞[1] 孟清[1] 

机构地区：[1]东华大学生物科学与技术研究所,中国上海201620

出　　处：《生命科学研究》2014年第1期28-32,49,共6页Life Science Research

基　　金：国家自然科学基金资助项目(31070698);国家高技术研究发展计划项目(863计划)(2006AA03Z451);上海市科委基础研究重点项目(10JC1400300);教育部博士点基金资助项目(20120075110007);教育部直属高校外聘专家-特色项目

摘　　要：蜘蛛丝是天然的生物材料,具有潜在的巨大应用价值。研究蜘蛛丝蛋白质的结构与功能,有助于破解蜘蛛丝蛋白质的成丝机理,为制备优良材料学性能的仿生蜘蛛丝纤维提供理论依据。以MiSp蜘蛛丝的重复区和C端非重复区蛋白多肽为研究对象,在不同pH值和离子条件下,在体外研究其二级结构与成丝的关系。CD图谱显示:表达纯化的重组蜘蛛丝蛋白R1R2在pH 7.5、6.5和5.5时二级结构相似,均为无规则卷曲,而R1R2CT则主要呈现为α螺旋构象;扫描电镜结果表明:在以上3种pH条件下,只有pH 5.5时R1R2和R1R2CT才形成重组丝纤维,R1R2CT纤维形态较平整,类似于天然蛛丝纤维形态,而R1R2丝纤维则呈条带状,表面粗糙。另外,氯化钠不利于形成形态平整的丝纤维。该成果为研究蛛丝蛋白的成丝机理奠定基础,也为制备仿生蛛丝蛋白纤维提供理论依据。In order to determine the structure and function of MiSp repetitive and nonrepetitive domains, R1R2 and R1R2CT were expressed recombinantly, after which secondary structures and protein-fibers for- mation were analyzed under different conditions. Through PCR and Bsa I digestion, R1R2 and R1R2CT modules of MiSp were cloned and expressed in Rosetta 2 （DE3） cells. After that the recombinant R1R2 and R1R2CT proteins were purified by Ni-NTA. CD spectra at room temperature showed R1R2 has similar sec- ondary structure （random coil） under pH 7.5, 6.5 and 5.5, however, the predominant secondary structure of R1R2CT in the same condition is a-helix. The results of SEM, which was applied to observe the assemble and fiber formation under different pH with or without NaC1, indicated that at pH 5.5 R1R2 and R1R2CT both can form fibers spontaneously： R1R2CT fibers are smooth and similar to native spider silks, while R1R2 fibers show crude fiber belt morphology. Furthermore, NaCl can give birth to fibers with crude mor- phology. The whole data show the secondary structure for R1R2 is random coil and α-helix for R1R2CT, in which CT might play an important role in stabilizing R1R2 secondary structures. Under pH 5.5, R1R2 and R1R2CT form fibers and NaCl can affect negatively fiber morphology. The results give clues to the proteins- silk formation mechanism research and are helpful to biomimic spider silks.

关 键 词：次壶腹腺丝蛋白(MiSp) 重组模块 Α螺旋 纤维 

分 类 号：Q785[生物学—分子生物学]