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目的构建DNA的G四链体抗体的原核表达质粒,利用BL21(DE3)菌表达此抗体,并进行纯化及鉴定。方法化学合成DNA的G四链体抗体的基因BG4,插入以p ET-26b(+)为骨架构建的载体p SANG10中,构建DNA的G四链体抗体表达载体p SANG10-BG4。以大肠杆菌BL21(DE3)为宿主菌进行该抗体的自诱导表达,渗透压裂解法收集此抗体,并经His亲和层析纯化,用SDS-PAGE及Western blot法鉴定此抗体,并在SW480结肠癌细胞中验证此抗体功能。结果 DNA的G四链体抗体表达载体经双酶切及基因测序鉴定构建成功。该抗体相对分子质量(Mr)为30 000~37 000,以可溶性的形式表达于BL21菌细胞间质,表达产物与目的蛋白大小一致。结论成功制备了DNA的G四链体抗体。
Objective To construct a prokaryotic expression plasmid for G quadruplex antibody of DNA and to express and purify the antibody using BL21 (DE3). Methods The gene G4 of G quadruplex antibody was chemically synthesized and inserted into the vector p SANG10 constructed with p ET-26b (+) as a backbone to construct the G quadruplex antibody expression vector p SANG10-BG4. This antibody was expressed in Escherichia coli BL21 (DE3) as a host strain. The antibody was collected by osmotic lysis and purified by His affinity chromatography. The antibody was identified by SDS-PAGE and Western blot. This antibody function was verified in colon cancer cells. Results The DNA G quadruplex antibody expression vector was successfully constructed by double enzyme digestion and sequencing. The relative molecular mass of the antibody (Mr) was 30 000 ~ 37 000, and was expressed in soluble form in the interstitial cells of BL21 strain. The expressed product showed the same size as the target protein. Conclusion The G quadruplex antibody of DNA was successfully prepared.