大腹园蛛管状腺丝重组蛋白的克隆表达及纺丝  

Expression and spinning of tubuliform silkfiber recombinant protein from Araneus ventricosus

作　　者：汪双祝 温睿 王康康 田露阳 孟清[1] 林瑛[1] WANG Shuangzhu;WEN Rui;WANG Kangkang;TIAN Luyang;MENG Qing;LIN Ying(Department of Bioengineering,College of Chemistry Chemical Engineering and Bioengineering,Donghua University,Shanghai,201620)

机构地区：[1]东华大学化学化工与生物工程学院,上海201620

出　　处：《工业微生物》2020年第2期15-21,共7页Industrial Microbiology

基　　金：上海市科学技术委员会自然科学基金项目(19ZR1471000);国家自然科学基金委面上项目(31470836)。

摘　　要：TuSp1蛋白(tubuliform spidroin 1)是管状腺丝(tubuliform silkfiber)的主要组成成分。管状腺丝作为蛛丝卵袋的外层包卵丝,其结构具有很好的耐腐蚀性和良好的力学性能。目前国内外对大腹园蛛TuSp1蛋白的研究很少,仅有一条基因序列的报道。本课题首次构建含大腹园蛛N端非重复结构域、重复单元以及C端非重复结构域的重组管状腺丝蛋白TuSp1 NT-Rp-CT,并经湿法纺丝获得重组蛋白丝纤维。重组蛋白液圆二色谱分析结果显示,pH由7.0降低到5.5的过程中,始终保持稳定的α-螺旋构象;重组蛋白丝纤维的傅里叶变换红外光谱结果显示,丝纤维中主要二级结构为β-折叠及β-转角;经扫描电镜观察发现,冻干的絮状重组蛋白能自组装成丝纤维,且表面光滑纤细;湿纺后的重组蛋白丝纤维直径较粗,但表面较平整均匀,具有类似天然管状腺丝的形态特征,这些为TuSp1蛋白的成丝机理及仿生纺丝研究提供了理论依据。Tubuliform spidroin 1(TuSp1)is the main constituent of tubuliform silk.As the outer layer of the egg sac,tubuliform silk has remarkable corrosion resistance and mechanical properties.At present,there are few studies on TuSp1 from Araneus ventricosus both at home and abroad,exept one gene sequence reported.This study is the first time to construct a recombinant tubuliform silk containing a non-repetitive N-terminal domain,a repetitive region and a non-repetitive C-terminal domain.And then recombinant spider silk fibers were obtained by wet spinning.The circular dichroism(CD)analysis of recombinant protein showed that the recombinant protein solution maintained its own stableα-helix during the pH reduction from 7.0 to 5.5;The results of fourier transform infrared spectroscopy showed that the main secondary structures in silk fibers wereβ-sheet andβ-turn.SEM observation results showed that the freeze-dried recombinant protein could self-assemble into silk-like fiber,and the surface was smooth and slender;the diameter of the wet-spun recombinant protein fiber was thicker,but the surface was smooth and uniform,which was similar to the morphological characteristics of natural tubuliform silk.This study provided certain theoretical basis for the subsequent studies on the mechanism of tubuliform silk TuSp1 and spinning conditions.

关 键 词：管状腺丝 原核表达 湿法纺丝 圆二色谱 傅里叶红外光谱 

分 类 号：TQ3[化学工程]