重组杂合蛛丝蛋白MiSpNT-PySpRp-MiSpCT的二级结构表征

Characterization of Secondary Structure of a Recombinant Hybrid Spider Silk MiSpNT-PySpRp-MiSpCT Protein

重组蛛丝纤维作为一种性能优异的生物材料,具有良好的生物相容性,在生物医学工程领域具有极大的潜在应用价值。已有研究表明,重组蛛丝蛋白可用作血管、神经导管及药物载体等,但其生物学功能仍有待研究。本研究以大腹园蛛基因组为模板,设计特异性引物,通过PCR扩增获得大腹园蛛梨状腺丝(piriform spidroin:Py Sp)一个完整重复区(Rp)编码序列;此Rp模块与Mi Sp NT/CT模块重组,构建微小型杂合蛛丝蛋白Mi Sp NT-Py SpRp-Mi Sp CT,成功在大肠杆菌BL21中高效表达,借助8 mol/L尿素裂解缓冲液进行变性纯化,得到纯度较高的杂合蛛丝蛋白Mi Sp NTPy SpRp-Mi Sp CT,产量约100 mg/L。CD图谱显示,Mi Sp NT-Py SpRp-Mi Sp CT蛋白质溶液主要以α-螺旋和无规卷曲形式存在,随着溶液p H值降低,部分α-螺旋向β-折叠转变;红外光谱显示,在自然成丝及冻干过程中,部分α-螺旋转化为β-折叠,符合天然蛛丝蛋白成丝过程的二级结构变化特征。本研究结果为今后获得具有天然蛛丝纤维优异性能的人工重组蛛丝纤维材料提供一种新的可能。

Recombinant spider silk,a potential excellent material,has biocompatibility and great potential application value in the field of biomedical engineering.Recent research has demonstrated that recombinant spider silk could be used for biomaterials such as vessels,nerve conduit and drug carrier.However,further studies are needed to elicit its biological function.In this study,specific primers were designed and Araneus ventricosus genome was used as a template to clone a complete repeat region（Rp）coding sequence of the Araneus ventricosus Py Sp.The Py Sp-Rp was cloned into a micro-hybrid spider silk protein Mi Sp NT-Py SpRp-Mi Sp CT construct which was successfully expressed in E.coli BL21.The hybrid spider silk protein Mi Sp NT-Py SpRp-Mi Sp CT with a relatively high purity was obtained by using 8 mol/L urea lysis buffer to conduct denaturation and purification.The expression level was relatively higher with about 100 mg/L.The CD spectra showed that the Mi Sp NT-Py SpRp-Mi Sp CT protein solution mainly contained α-helix and random coil forms,and parts of α-helixes transited to β-sheet with the decrease of the p H value.The infrared spectra showed that the Mi Sp NT-Py SpRp-Mi Sp CT protein of α-helixestransited to β-sheet in a natural silk forming and freeze-drying process.The results were consistent with the characterization of secondary structure during silk forming process.This study will provide a new possibility to obtain artificial recombinant spider silk fiber material with excellent mechanical properties of natural silk fiber in future.