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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1.2mg/ml。制备了该蛋白的多克隆抗体,ELISA表明多克隆抗体效价为1:32000,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。
Objective To express and purify the modified TAT-VP3 fusion protein and prepare its polyclonal antibody. Methods The prokaryotic expression vector PGEX-6P-1 / TAT-VP3 was expressed in E.coli Rosetta (DE3). The purified protein was purified by GST-labeling and the protein was excised by PreScission Protease. The purified protein was used to immunize New Zealand white rabbits , A polyclonal antibody to the modified TAT-VP3 fusion protein was obtained. The titer was detected by ELISA and the specificity was identified by Western blotting. Results The recombinant TAT-VP3 fusion protein was induced and purified with a purity of more than 90% and a protein concentration of 1.2 mg / ml. The polyclonal antibody of this protein was prepared. The ELISA showed that the titer of polyclonal antibody was 1: 32000, and the specificity of polyclonal antibody was proved by Western blotting. Conclusion The recombinant TAT-VP3 fusion protein was successfully purified and a polyclonal antibody with high titer and high specificity was prepared, which laid the foundation for further study on antitumor activity and mechanism of TAT-VP3 fusion protein in vitro and in vivo.