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目的构建融合蛋白PEP-1-VP3的原核表达质粒,并进行诱导表达和蛋白纯化。方法通过PCR方法自PGEX-6P-1/TAT-VP3质粒中扩增VP3基因,运用靶向克隆酶,将VP3基因插入到线性载体p ET15b-PEP-1中,构建重组表达质粒p ET15b-PEP-1-VP3,经测序证实构建成功后转化E.coli Rosetta,表达PEP-1-VP3融合蛋白,经Ni-NTA树脂亲和层析,进行SDS-PAGE及Western Blot分析鉴定。结果成功构建PEP-1-VP3融合蛋白原核表达载体,表达并纯化了17.6k D的PEP-1-VP3融合蛋白。结论所构建的PEP-1-VP3融合蛋白为体内抗肿瘤研究提供了理论基础。
Objective To construct a prokaryotic expression plasmid of fusion protein PEP-1-VP3 and induce its expression and protein purification. Methods The VP3 gene was amplified by PCR from the plasmid pGEX-6P-1 / TAT-VP3. The target gene was cloned and inserted into the linear vector pET15b-PEP-1. The recombinant plasmid p ET15b-PEP -1-VP3. After successful sequencing, E.coli Rosetta was transformed into E.coli Rosetta to express PEP-1-VP3 fusion protein. The fusion protein was identified by SDS-PAGE and Western Blot analysis by Ni-NTA resin affinity chromatography. Results The prokaryotic expression vector of PEP-1-VP3 fusion protein was successfully constructed and the 17.6kD PEP-1-VP3 fusion protein was expressed and purified. Conclusion The constructed PEP-1-VP3 fusion protein provides a theoretical basis for in vivo anti-tumor research.