莱茵衣藻ChR2多克隆抗体的制备

Preparation of Polyclonal Antibodies Against Chlamydomonas reinhardtii ChR2

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∶25 600；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93 000），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。

Abstract:

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 1:25 600. The target band (relative molecular mass 93 000) 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.