苏云金杆菌Cry2Ab可溶蛋白的原核表达及多克隆抗体的制备  

Prokaryotic Expression of the Soluble Cry2Ab Protein from Bacillus thuringiensis and Preparation of the Polyclonal Antibody Against Cry2Ab

作　　者：邵恩斯[1] 林莉[1] 关雄[1] 

机构地区：[1]福建农林大学生物农药与化学生物学教育部重点实验室,福州350002

出　　处：《农业生物技术学报》2013年第1期106-111,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31071745);高等学校博士学科点专项科研基金(No.20093515110010);福建省科技厅产学研重点项目(No.2011N5003)

摘　　要：苏云金杆菌Cry2Ab蛋白是一类对鳞翅目昆虫有特异性毒性作用的毒素蛋白,已广泛应用于针对鳞翅目害虫的防治之中。依据苏云金杆菌cry2Ab基因序列设计一对全长引物,从苏云金杆菌(Bacillus thuringiensis)WB9菌株总DNA中克隆出cry2Ab基因全序列,构建Cry2Ab-PK表达载体,将获得的Cry2Ab-PK阳性克隆进行原核诱导表达,获得约90kD的Cry2Ab-GST融合蛋白,经批量纯化并切除GST标签后获得可溶Cry2Ab蛋白,约65kD。利用纯化Cry2Ab可溶蛋白免疫新西兰雄性大白兔(Oryctolagus cuniculus),制备Cry2Ab兔源多克隆抗血清,通过间接ELISA法测定Cry2Ab抗血清效价超过1:150000。Western blot测定结果表明,制备的Cry2Ab兔源抗血清能特异性识别Cry2Aa及Cry2Ab抗原蛋白,不能识别Cry1Ab及Cry3Aa抗原蛋白。研究结果对深入研究Cry2A毒素蛋白作用机理及毒素与受体间互作关系提供了技术支持。Cry2Ab toxin of Bacillus thuringiensis is a toxic protein, which is wildly used in controlling lepidopteran pest in agricultural production. In this research, the cry2A b gene （1 914 bp） was amplified from total DNA ofB. thuringiensis WB9 strain by a pair of primer designed by the full-length sequence of published cry2A b gene. Then, cry2A b was ligated with linearized pGEX-KG vector by restriction enzyme BamH I and Xho I for the construction of cry2Ab-pk expression vector. The soluble Cry2Ab-GST fusion protein （approximately 90 kD） was obtained after transferring Cry2Ab-PK expression vector into Escherichia coli BL21 （DE3） and then inducing by 0.8 mmol/L IPTG at 16℃ for 36 h. Total soluble protein was purified by batch purification and GST tag was removed by the use of prescission protease to obtain soluble Cry2Ab protein （approximately 65 kD）. Polyclonal antibody against Cry2Ab was produced by immunizing the purified Cry2Ab to New Zealand white rabbit （Oryctolagus cuniculus） after three times of intramuscular injection and one time of intravenous injection. The titter of antibody was over 1：150 000, measured by indirect ELISA.Specificity of the prepared antibody was determined by Western blot, showing that the polyclonal antibody against the Cry2Aa or Cry2Ab protein was positive and the antibody against CrylAb or Cry3Aa protein was negative. These results indicated that antibody against Cry2Ab protein can specifically identify Cry2A protein but cannot identify other three domains Cry protein including CrylAb and Cry3Aa. These results will provide technical support for further study of Cry2A toxins mechanism and the interaction between Cry2A toxins and its receptors.

关 键 词：苏云金杆菌 CRY2AB 可溶蛋白 多克隆抗体 

分 类 号：S432.23[农业科学—植物病理学]