DNA-PKcs单链抗体DPK3-scFv的原核表达纯化及生物学作用研究

摘要

目的 原核表达和纯化DNA依赖性蛋白激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)单链抗体DPK3-scFv,观察其透入细胞及干扰辐射诱发DNA-PKcs磷酸化修饰的生物学作用.方法 PCR扩增DPK3-scFv基因,插入到带有His标签的原核表达载体pET28a,重组质粒转化工程茵BL21、优化表达.免疫荧光显微镜和蛋白免疫印迹观察DPK3-scFv透膜进入HeLa细胞及对电离辐射诱发靶分子DNA-PKcs的磷酸化修饰的影响.结果 成功构建单链抗体表达载体pET28a-DPK3-scFv,在2xYT培养基(16 g胰蛋白胨、5 g酵母提取物和lO g NaCI溶于1 L双蒸水中,高压灭茵)、20℃温度、O.2 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)条件下诱导可溶性表达,进一步获得了纯化单链抗体.DPK3-scFv能体外抑制DNA-PKcs激酶活性,纯化的DPK3-scFv可跨膜进入细胞内,并与DNA-PKcs共定位在细胞核.DPK3-seFv对γ射线照射HeLa细胞中DNA-PKcs及其S2056位点(pS2056)的自磷酸化具有显著抑制作用.结论 通过优化条件获得了可溶性原核表达的DPK3-scFv单链抗体,具有抑制其靶分子DNA-PKcs的磷酸激酶活性.%Objective To study prokaryotic expression, purification of the single-chain antibody DPK3-scFv against DNA-PKcs, and to observe its interference with the phosphorylation of DNA-PKcs in response to ionizing radiation. Methods DPK3-scFv gene produced by PCR was inserted into the prokaryotic expression vector pET28a with His tag. The re-combinant plasmids were transformed into bacteria strain BL21, and the expression conditions were optimized. The transmembrane and biological activity of DPK3-scFv were assessed by immunofluorescence microscope observation and Western immunoblotting. Results The recombinant prokaryotic expression vector of pET28a-DPK3-scFv was constructed. Optimized expression of soluble DPK3-scFv was obtained in E. Coli BL21 cultured in 2xYT medium with 0. 2 mmol/L isopropyl-β-D-thiogalactopyranoside( IPTG ) induction at 28℃. The purified DPK3-scFv could effectively inhibit the kinase activity of DNA-PKcs in vitro. The results of immunofluorescence observation and Western immunoblotting indicated that this recombinant scFv could enter cells and colocalize mainly with DNA-PKcs in the nuclei. DPK3-scFv significantly inhibited DNA-PKcs expression and abolished the phosphorylation of DNA-PKcs/pS2056 in response to γ-ray irradiation. Conclusion The soluble expression of DPK3-scFv antibody is obtained in E. Coli BL21. DPK3-scFv can enter nuclei and has biological effect on inhibiting the kinase activity of DNA-PKcs.