马铃薯蔗糖磷酸合成酶StSPS1的原核表达及抗体制备

Prokaryotic Expression and Antibody Preparation of Potato Sucrose Phosphate Synthase StSPS1

为了对马铃薯蔗糖磷酸合成酶(SPS)编码蛋白StSPS1进行原核表达及多克隆抗体制备。从四倍体马铃薯品种川芋10号的块茎中克隆出了StSPS1基因,该基因编码区全长为3165 bp,编码蛋白的长度为1055 aa。随后基于构建的His标签融合表达载体PET30a-StSPS1,进行了StSPS1蛋白的诱导、变性、纯化、复性及兔免疫试验。结果发现,StSPS1蛋白分子量约为119.62 ku,在可溶性上清中的表达极少,主要在不溶的沉淀中表达,最佳的诱导条件为37℃下用0.5或1.0 mmol/L的IPTG诱导4 h。由于StSPS1为包涵体蛋白,故对其进行包涵体变性处理,并利用His标签纯化出了与目的条带大小相符的蛋白,同时通过His抗体进行了蛋白质免疫印迹(WB)试验,发现在119.62 ku处检测到目标条带,说明StSPS1包涵体蛋白纯化成功。最后,通过将透析复性后的StSPS1蛋白注射进兔子皮下组织中,成功免疫出2个StSPS1的抗体,经过WB鉴定发现2个抗体均能在抗原和川芋10号叶片的总蛋白中杂出目标条带。综上,对马铃薯StSPS1蛋白进行了诱导及纯化,并成功制备了StSPS1蛋白的兔多克隆抗体。

To express the protein StSPS1 encoding potato sucrose phosphate synthase(SPS)in E.coli and prepare polyclonal antibodies.The StSPS1 gene was cloned from the tubers of tetraploid potato variety Chuanyu 10,with a total coding region of 3165 bp and a protein length of 1055 aa.Subsequently,based on the constructed His tag fusion expression vector PET30a-StSPS1,the protein induction,denaturation,purification,renaturation,and rabbit immune tests of StSPS1 protein were carried out.The results showed that the molecular weight of StSPS1 protein was approximately 119.62 ku,and its expression was minimal in soluble supernatant,mainly in insoluble precipitates.The optimal induction conditions were induced with 0.5 or 1.0 mmol/L IPTG at 37℃for 4 h.Due to StSPS1 being an inclusion body protein,it was subjected to inclusion body denaturation and purified using His tags to match the size of the target band.At the same time,a protein immunoblotting(WB)test was performed using His antibodies,and the target band was detected at 119.62 ku,indicating the successful purification of StSPS1 inclusion body protein.Finally,by injecting the dialyzed and refolded StSPS1 protein into the subcutaneous tissue of rabbits,two antibodies against StSPS1 were successfully immunized.After WB identification,it was found that both antibodies could hybridize target bands in the antigen and the total protein of leaves in Chuanyu 10.In summary,potato StSPS1 protein was induced and purified,and rabbit polyclonal antibodies against StSPS1 protein were successfully prepared.