改良型TAT-VP3融合蛋白的原核表达纯化及多克隆抗体的制备

目的 表达、纯化改良型TAT-VP3融合蛋白,并制备其多克隆抗体.方法 利用原核表达载体PGEX-6P-1/改良型TAT-VP3在大肠杆菌Rosetta(DE3)中表达,经GST标签纯化树脂纯化蛋白,并用PreScission Protease酶切除标签,以纯化的蛋白免疫新西兰大白兔,获得改良型TAT-VP3融合蛋白的多克隆抗体.用ELISA进行效价检测,Western blotting鉴定其特异性.结果 诱导表达并纯化了改良型TAT-VP3融合蛋白,纯度大于90%,蛋白浓度为1.2 mg/ml.制备了该蛋白的多克隆抗体,ELISA表明多克隆抗体效价为1∶32 000,Western blotting证明多克隆抗体的特异性良好.结论 成功纯化出改良型TAT-VP3融合蛋白,并制备出高效价、高特异性的多克隆抗体,为进一步研究该蛋白的体内外抗肿瘤活性及作用机制奠定了基础.

Expression, purification of improved TAT-VP3 fusion protein and preparation of its polyclonal antibody

Objective To express and purify improved TAT-VP3 fusion protein and to prepare its polyclonal antibody.Methods PGEX-6P-1/improved TAT-VP3 was expressed in E.coli Rosetta(DE3).The expressed protein was purified by GST Resin.The label of protein was cut by PreScission Protease.The purified protein was used to immunize New Zealand rabbits for preparing polyclonal antibody.The titer of the polyclonal antibody was detected by ELISA.The specificity was tested by Western blotting.Results The purity of improved TAT-VP3 fusion protein was higher than 90%,and its concentration reached 1.2 mg/ml.Its polyclonal antibody showed a titer of 1:3 2000 by ELISA.Specificity was confirmed well by Western blotting.Conclusions The improved TAT-VP3 is expressed and purified successfully.The polyclonal antibody is also of high titer and specificity.Our study provided the foundation for further research of its antitumor activity and mechanism in vitro and in vivo.