胶原蛋白作为细胞外基质中关键的结构性和功能性蛋白,其重组表达形式相比于天然提取具备无毒性、低免疫原性、高溶解度和质量一致性等优势。目前重组胶原蛋白通过光交联方式制备水凝胶的研究在序列方面仍旧存在诸多难题。因此该文探究不同长度重组胶原蛋白分子通过甲基丙烯酰化后光交联对水凝胶性能的影响。基于前期的研究结果,实验选用2种重组胶原蛋白分子V-P5BP5和V-P10BBP10作为研究对象,其中V-P5BP5序列为V-P10BBP10序列长度的0.5倍。研究结果表明,P5BP5和P10BBP10在大肠杆菌中成功表达。圆二色光谱测定显示P5BP5和P10BBP10的分子椭圆率分别为3.17和2.62,Tm分别为44、49 ℃。重组胶原蛋白经过甲基丙烯酰化后,核磁共振氢谱在5.4、5.8 ppm附近出现新的特征峰,红外光谱在1 130、1 730 cm-1附近出现新的特征吸收峰,证实甲基丙烯酸酐的成功插入。通过光交联方式制备水凝胶,微流变实验显示P10BBP10甲基丙烯酰化重组胶原蛋白在频率为2.72 rad/s时G′=G″,低频下状态为水凝胶,高频下状态为溶液,宏观结果呈现形成具有自支撑性能的三维网络结构。而P5BP5甲基丙烯酰化重组胶原蛋白仅形成了黏性溶液。该研究通过将不同序列长度的胶原蛋白分子进行甲基丙烯酰化反应,为开发光交联制备水凝胶在序列长度设计领域提供依据,为组织工程和生物医用材料的构建奠定了理论基础。
As a key structural and functional protein in the extracellular matrix, collagen’s recombinant expression form has the advantages of non-toxicity, low immunogenicity, high solubility, and quality consistency compared with natural extraction.Currently, the preparation of hydrogels from recombinant collagen via photo-crosslinking still encounters many challenges in terms of sequence design.Therefore, this paper investigated the effects of photo-crosslinking of recombinant collagen of different lengths on hydrogel properties after methacrylylatelation.Based on the results of previous research, two recombinant collagen molecules, V-P5BP5 and V-P10BBP10, were selected as the research objects, in which the V-P5BP5 sequence was 0.5 times the length of the V-P10BBP10sequence.The results showed that P5BP5 and P10BBP10 were successfully expressed in Escherichia coli.Circular dichroic spectroscopy assays showed molecular ellipticity of P5BP5 and P10BBP10 of 3.17 and 2.62, respectively, and Tm of 44 and 49 ℃, respectively.In methacrylylated recombinant collagen, new characteristic peaks appeared in nuclear magnetic resonance hydrogen spectroscopy around 5.4 and 5.8 ppm, and new characteristic absorption peaks around 1 130 and 1 730 cm-1 in infrared spectroscopy, confirming the successful insertion of methacrylic anhydride.Microrheological experiments showed that P10BBP10 methylacrylated recombinant collagen was G′=G″ at a frequency of 2.72 rad/s, a hydrogel at low frequency, and a solution at high frequency.P5BP5 methylacrylated recombinant collagen only forms a viscous solution.This study provides a basis for the development of photo-crosslinked hydrogels in the field of sequence length design by performing methacrylyl reactions of collagen molecules with different sequence lengths, and lays a theoretical foundation for the construction of tissue engineering and biomedical materials.
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