拟南芥叶绿体发育必需蛋白PAC的原核表达及多克隆抗体制备

Prokaryotic expression and preparation of polyclonal antibody for essential protein PAC of Arabidopsis chloroplast

【目的】将拟南芥叶绿体发育必需蛋白PAC进行大肠杆菌原核表达和纯化,并免疫家兔,获得针对PAC蛋白的多克隆特异性抗体,为进一步研究PAC蛋白在叶绿体发育中的功能提供保证。【方法】将编码拟南芥PAC蛋白的cDNA克隆至原核表达载体pET-28a中,转化BL21(DE3)表达菌,经IPTG诱导后,对诱导产生的PAC蛋白进行镍柱亲和纯化和SDS-PAGE割胶纯化,并将纯化的PAC蛋白通过皮下注射免疫家兔,利用分离得到的抗血清,针对拟南芥野生型、PAC敲除突变系pac-1和PAC过表达系PACOE的总蛋白进行免疫印迹检测。【结果】成功构建了拟南芥基因PAC的原核表达载体pET-28a-PAC,在37℃、1mmol/L IPTG诱导4h的大肠杆菌细胞中,可检测到分子质量为38.9ku的重组蛋白,其大部分以可溶形式存在。用纯化诱导后的重组蛋白免疫家兔获得PAC抗血清,该抗血清能够有效地检测出1ng原核表达的PAC重组蛋白,并在植物样品中检测出1条分子质量约为35ku的条带。【结论】成功制备了PAC蛋白的多克隆抗体,可用于植物中PAC蛋白含量的检测。

【Objective】The essential protein PAC of Arabidopsischloroplast was purified and expressed in E.coli before being injected into rabbits as antigen to generate specific polyclonal antibody.【Method】PAC cDNA was cloned into prokaryotic expression vector pET-28 a,and the recombinant vector was transformed into E.coli BL21（DE3）.After IPTG induction,induced PAC was purified via Nickel affinity chromatography,followed by electrophoretic elution from SDS-PAGE gel slices.Purified PAC protein was used to immunize rabbits,and anti-PAC antiserum was obtained and tested against total proteins from Arabidopsis,including the wild type,the PAC knockout line and PAC overexpression lines with Western blot.【Result】The prokaryotic expression vector pET-28a-PAC was constructed successfully and expressed in E.coli BL21（DE3）induced with 1mmol/L IPTG.A recombinant protein with molecular weight of 38.9ku was detected.The anti-PAC antiserum was efficient to detect 1ng of PAC antigen.A specific band withmolecular weight of-35ku corresponding to PAC in total protein of Arabidopsis was also detected.【Conclusion】The specific antiserum against PAC protein was prepared successfully,which can be used for detection of PAC in chloroplasts.