摘要
【目的】制备一支莱茵衣藻(Chlamydomonas reinhardtii,C.reinhardtii)光敏通道蛋白2(ChR2)多克隆抗体,研究ChR2在鞭毛内通过感应光信号来影响细胞趋光性的应答机制。【方法】将构建的表达载体pET-28a(+)-chr2转化至大肠杆菌(Escherichia coli,E.coli)BL21(DE3)感受态细胞,诱导表达6×His-ChR2融合蛋白,用镍柱对其进行亲和纯化。以此融合蛋白作为抗原进行动物免疫,产生相应抗体,再采用Protien A处理抗血清,使得抗体富集,获得纯度较高的莱茵衣藻ChR2多克隆抗体。通过免疫印迹检测莱茵衣藻ChR2多克隆抗体的特异性,并通过免疫荧光试验来确定ChR2在鞭毛中的位置。【结果】6×His-ChR2融合蛋白纯化结果表明其纯度>85%;间接ELISA法检测莱茵衣藻ChR2多克隆抗体的效价为1∶25600;在野生型莱茵衣藻CC-125细胞的全蛋白质提取物中出现了目的条带(相对分子质量93000),而在突变型莱茵衣藻CC-5499(chr1和chr2双基因敲除的突变体)细胞的全蛋白质提取物中未出现目的条带,说明所得到的莱茵衣藻ChR2多克隆抗体特异性较高;ChR2定位于莱茵衣藻细胞的眼点和鞭毛。【结论】该研究为后续探索ChR2在鞭毛内通过感应光信号影响细胞趋光性的机制奠定了基础。
[Objective]This study prepared the polyclonal antibodies against Chlamydomonas reinhardtii(C.reinhardtii)channelrhodopsin 2(ChR2)to study the mechanism by which ChR2 in flagella guides cell phototaxis by sensing light signals.[Method]The constructed expression vector pET-28a(+)-chr2 was transformed into Escherichia coli BL21(DE3)competent cells to induce the expression of the 6×His-ChR2 fusion protein,which was then purified through a nickel column.The fusion protein was used as an antigen to immunize animals for the production of corresponding antibodies.The antiserum was treated with Protien A to enrich the antibodies,and thus high-purity polyclonal antibodies against C.reinhardtii ChR2 were obtained.Western blot was employed to assess the specificity of the prepared antibodies,and immunofluorescence was used to determine the localization of ChR2 in flagella.[Result]The purification results of the 6×His-ChR2 fusion protein demonstrated a purity exceeding 85%after purification.The indirect ELISA results showed that the titer of the polyclonal antibodies against C.reinhardtii ChR2 reached 1:25600.The target band(relative molecular mass 93000)appeared in electrophoresis of the whole protein extract of wild-type C.reinhardtii CC-125 cells but did not appear in that of the whole protein extract of mutant C.reinhardtii CC-5499(a mutant with knockout of both chr1 and chr2)cells,indicating that the obtained polyclonal antibodies had high specificity.The localization of ChR2 was in the eyespots and flagella of C.reinhardtii cells.[Conclusion]The findings lay a foundation for subsequent studies on the mechanism by which ChR2 in flagella guides cell phototaxis by sensing light signals.
作者
张媛媛
刘雁霞
樊振川
ZHANG Yuanyuan;LIU Yanxia;FAN Zhenchuan(College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457,China;National International Science and Technology Cooperation Institute of Health Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;State Key Laboratory of Food Nutrition and Safety,Tianjin University of Science and Technology,Tianjin 300457,China)
出处
《食品与生物技术学报》
北大核心
2025年第2期63-70,共8页
Journal of Food Science and Biotechnology
基金
国家自然科学基金面上项目(32070698)
国家自然科学基金青年基金项目(32200558)。
关键词
莱茵衣藻
ChR2
原核表达
多克隆抗体
Chlamydomonas reinhardtii
ChR2
prokaryotic expression
polyclonal antibody
