莱茵衣藻光敏通道蛋白质ChR1的原核表达纯化及多克隆抗体制备

Prokaryotic Expression, Purification, and Polyclonal Antibody Preparation of Chlamydomonas reinhardtii Channelrhodopsin 1

摘要:

【目的】制备一支高特异性的莱茵衣藻(Chlamydomonas reinhardtii，C. reinhardtii)光敏通道蛋白质1（channelrhodopsin 1，ChR1）抗体，研究ChR1调控细胞趋/避光性的机制。【方法】构建重组表达载体pET-28a（+）-chr1并转入大肠杆菌（Escherichia coli，E.coli）BL21（DE3）感受态细胞，经IPTG诱导表达6×His∷ChR1融合蛋白，后经镍柱亲和纯化后免疫新西兰大白兔获得ChR1抗血清，利用protein A纯化对ChR1抗血清进行IgG亚型抗体富集获得ChR1抗体，免疫印迹检测ChR1抗体特异性，并通过免疫荧光确定ChR1在细胞中的定位。【结果】利用间接ELISA法测定ChR1的抗血清效价为1∶102 400；纯化获得的ChR1抗体通过免疫印迹检测到CC-125细胞中含有ChR1条带且无非特异性条带，表明抗体特异性高；免疫荧光试验结果显示，ChR1在细胞眼点位置有明显定位且无非特异性染色，表明制备的ChR1多克隆抗体适用于免疫荧光试验。【结论】作者成功实现了莱茵衣藻ChR1的原核表达纯化和多克隆抗体的制备，为ChR1在莱茵衣藻中的定位和调控趋/避光的分子机制研究奠定了重要基础。

Abstract:

[Objective] This study aims to prepare a highly specific antibody against channelrhodopsin-1 (ChR1) from Chlamydomonas reinhardtii (C. reinhardtii) and investigate its regulatory mechanisms in cellular phototaxis and negative phototaxis.[Method] The recombinant expression vector pET-28a(+)-chr1 was constructed and transformed into Escherichia coli (E. coli) BL21 (DE3) competent cells. The expression of the fusion protein 6×His∷ChR1 was induced with IPTG, and the obtained protein was purified by nickel-affinity chromatography. The purified protein was used to immunize New Zealand white rabbits for the production of ChR1 antiserum. ChR1-specific IgG antibodies were enriched from the antiserum through protein A purification. The antibody specificity was assessed by immunoblotting, and the subcellular localization of ChR1 was analyzed by immunofluorescence.[Result] The results of indirect ELISA indicated that the ChR1 antiserum had a titer of 1∶102 400. Immunoblotting with the purified ChR1 antibody identified a distinct band corresponding to endogenous ChR1 in CC-125 cells, with no nonspecific band observed, thereby confirming the high specificity of the antibody. Immunofluorescence staining revealed clear localization of ChR1 at the eyespot and specific staining, demonstrating the suitability of the antibody for immunofluorescence assays.[Conclusion] The prokaryotic expression, purification, and polyclonal antibody preparation of ChR1 were achieved. This study lays a crucial foundation for delving into the localization of ChR1 in C. reinhardtii and the molecular regulatory mechanisms of ChR1 in phototaxis and negative phototaxis.