小麦自噬标志分子ATG6的原核表达及其抗血清制备

Prokaryotic expression of recombinant ATG6 from wheat variety L_(10),a molecular marker of autophagy,and development of its antiserum

克隆小麦(Triticum aestivum L.)品种‘洛夫林10’(L10)中的ATG6(动物同源物为Beclin1)基因,并构建该基因的原核表达载体,在大肠杆菌中诱导表达融合蛋白,经纯化后制备其兔抗血清。结果表明:从小麦品种L10中克隆获得1个ATG6的片段,长为1326 bp,利用所构建的大肠杆菌表达载体,经IPTG诱导,实现了对ATG6片段在原核系统的特异性表达。经蛋白纯化后,制备了其兔抗血清并通过Western blot鉴定了其特异性。ATG6抗体制备的成功,为小麦中ATG6基因和自噬功能的研究提供了研究基础。

Molecular cloning of ATG6 from wheat cultivar L10,whose prokaryotic expression vector was then constructed and expressed in Escherichia coli rosetta-gami B（DE3）.The rabbit antiserum against ATG6 was prepared and characterized finally.The ATG6 was amplified from wheat variety L10 by PCR and then cloned into the pMD19-T vector,one of which was subcloned into the expression vector.The recombinant plasmid was identified by sequencing and digestion of restriction enzymes.The fusion protein was finally expressed by IPTG-induction in host bacteria-E.coli rosetta-gami B（DE3） and detected by SDS-PAGE.The rabbit anti-ATG6 antibody was prepared and detected by western blot analysis.The ATG6 was obtained partly and successfully expressed in the prokaryotic expression system.The rabbit anti-ATG6 antibody was prepared and detected by western blot.The experiment offered foundation to study autophagy and function of ATG6 gene in wheat.