莱茵衣藻纤毛内运送蛋白IFT70的原核表达、纯化及其多克隆抗体的制备

Prokaryotic expression and purification of Chlamydomonas reinhardtii intraflagellar transport protein 70 and preparation of its polyclonal antibody

目的原核表达及纯化莱茵衣藻纤毛内运送蛋白IFT70,并制备其多克隆抗体。方法以质粒p GAD-ift70(含莱茵衣藻ift70基因N-末端编码380个氨基酸的核酸序列)为模板,分别构建带有6×His和MBP标签的原核表达质粒p ET-28A-ift70和p MAL-C2X-ift70,转入E.coli BL21(DE3),经IPTG诱导表达6×His-IFT70和MBP-IFT70融合蛋白,并进行亲和层析。将纯化的MBP-IFT70融合蛋白免疫2只新西兰大白兔,经耳动脉采血,分离血清,ELISA法测定抗体效价,经两步亲和纯化后进行Western blot及免疫荧光法分析。结果经双酶切及测序鉴定证明原核表达质粒p ET-28A-ift70和p MAL-C2X-ift70构建正确。6×His-IFT70和MBP-IFT70融合蛋白的相对分子质量分别为40 000和80 000,纯化后纯度可达90%。家兔1及家兔2抗血清的效价分别为>128 000及>256 000,纯化的抗血清可与莱茵衣藻IFT70蛋白发生特异性结合,于相对分子质量约70 000处可见特异性结合条带,可与野生型莱茵衣藻CC-125纤毛上的IFT70蛋白发生特异反应,荧光显微镜下可见红色荧光。结论成功制备了抗莱茵衣藻IFT70的多克隆抗体,为IFT70在IFT和纤毛组装中作用的深入研究奠定了基础。

Objective To express Chlamydomonas reinhardtii intraflagellar transport protein 70（IFT70） in prokaryotic cells, purify the expressed product and prepare its polyclonal antibody. Methods The 6 ×His-and MBP-tagged prokaryotic expression plasmids p ET-28A-ift70 and p MAL-C2X-ift70 were constructed by inserting the c DNA sequence encoding the 380 amino acids at N-terminus of C. reinhardtii IFT70 into the p ET-28A（＋） and p MAL-C2 X vectors respectively, and transformed to E. coli BL21（DE3）for expression under induction of IPTG. Fusion proteins 6 × His-IFT70 and MBP-IFT70 were expressed and purified by affinity chromatography. Two New Zealand white rabbits were immunized with purified MBP-IFT70, of which the serum samples were collected and determined for antibody titer, then purified by two step affinity chromatography and analyzed by Western blot and IFA. Results Restriction analysis and sequencing proved that recombinant plasmids p ET-28a-ift70 and p MAL-C2X-ift70 were constructed correctly. The relative molecular masses of fusion proteins 6 × His-IFT70 and MBP-IFT70 were 40 000 and 80 000, while the purities were 90%. The titers of antisera of two immunized rabbits were more than 128 000 and more than 256 000 respectively. The purified antisera showed specific binding band to C. reinhardtii IFT70, with a relative molecular mass of about 70 000. However, the antisera showed specific reaction with IFT70 on the cilia of wild C. reinhardtii CC-125, with red f luorescence under fluorescent microscope. Conclusion The polyclonal antibody against IFT70 of C. reinhardtii was prepared successfully,which laid a foundation of further study on the role of IFT70 in the assembly of IFT and cilia.