重组类胶原蛋白的表达纯化及性质研究  

Study on expression,purification,and properties of recombinant type I human-like collagen

作　　者：刘迪 郭君慧 李其昌 尹巧玲 次佩松 樊李红 谢浩 LIU Di;GUO Junhui;LI Qichang;YIN Qiaoling;CI Peisong;FAN Lihong;XIE Hao(School of Chemistry,Chemical Engineering and Life Science,Wuhan University of Technology,Wuhan 430070,China)

机构地区：[1]武汉理工大学化学化工与生命科学学院,湖北武汉430070

出　　处：《食品与发酵工业》2025年第6期127-133,共7页Food and Fermentation Industries

基　　金：国家自然科学基金国际(地区)合作与交流项目(5181101987)。

摘　　要：天然组织中提取的胶原蛋白存在生物相容性的问题,而目前报道的重组胶原热稳定性及三螺旋水平较差。为此该研究选择毕赤酵母GS115作为异源表达菌株,诱导表达人I型胶原蛋白α1链部分胶原域片段。在C端添加T4 fibritin促折叠序列,利用圆二色谱热变温实验研究T4 fibritin对胶原热稳定性和三螺旋水平的影响,通过细胞增殖—毒性检测和自由基清除率评估了重组类胶原的生物活性。结果显示,含有T4 fibritin的重组类胶原1A1C热变性温度提高4℃,且热变温之后胶原能够复性。类胶原1A1和1A1C都表现出良好的生物相容性,在质量浓度5μg/mL时,类胶原1A1C能够显著促进成纤维细胞增殖。在质量浓度3 mg/mL时,两种方法测得的类胶原自由基清除率分别达到19.8%和40.0%。该研究利用基因工程方法重组表达了具有良好生物相容性的类胶原蛋白,通过融合端肽的方法使得到的类胶原热变性温度提高到39.9℃,高于人体温度37℃,具有较高应用潜力。The biocompatibility of collagen extracted from tissue poses a challenge,and the thermal stability and triple helix level of the reported recombinant collagen have been found to be suboptimal.Therefore,Pichia pastoris GS115 was chosen as the heterologous expression strain for inducing the expression of a fragment from the collagen domain of human type I collagenα1 chain.The T4 fibritin promoter sequence was added at the C-terminus.The impact of T4 fibritin on the thermal stability and triple helix level of collagen was investigated through circular dichroism thermal denaturation experiments.The bioactivity of the recombinant collagen was evaluated by cell proliferation-toxicity assay and free radical scavenging rate analysis.Results demonstrated that inclusion of T4 fibritin in recombinant collagen 1A1C increased its thermal denaturation temperature by 4℃,with subsequent regeneration after denaturation.Both human-like collagen 1A1 and 1A1C showed good biocompatibility.At 5μg/mL,human-like collagen 1A1C could promote the proliferation of fibroblasts significantly.Moreover,at 3 mg/mL,the scavenging rates of human-like collagen free radicals measured by two methods were 19.8%and 40.0%,respectively.This study successfully expressed biocompatible recombinant collagen using genetic engineering techniques.The thermal denaturation temperature of collagen was increased to 39.9℃by fusion of terminal peptide,which was higher than the human body temperature of 37℃,and have great potential for various applications.

关 键 词：重组类胶原 毕赤酵母 圆二色谱 成纤维细胞 

分 类 号：TS201.21[轻工技术与工程—食品科学]