双特异纳米抗体的融合表达、纯化及活性分析

Fusion expression,purification and activity of recombinant bispecific nano-antibody

目的融合蛋白表达癌胚抗原(carcinoembroynic antigen,CEA)/CD16双特异纳米抗体,并对其进行纯化及鉴定。方法将CEA/CD16基因克隆至pGEX4T1载体,构建重组表达质粒,转化E.coli BL21(DE3),经IPTG诱导表达。表达蛋白GST-CEA/CD16经GST亲和层析分离纯化、TEV酶切及Ni-NTA和Q-Sepharose FF纯化,获得目的蛋白,并对目的蛋白进行活性分析。结果重组表达质粒经双酶切及测序鉴定,证明构建正确。表达的融合蛋白相对分子质量约为58 000,主要以可溶性形式存在于菌体裂解上清中。融合蛋白经纯化后获得目的蛋白产率为5 mg/L,并具有介导NK细胞杀伤CEA阳性细胞的能力,且呈剂量依赖性。结论成功表达了GSTCEA/CD16融合蛋白,纯化后表达产率较高,且具有较好的生物学活性,表明GST融合表达方式应用于双特异抗体具有可行性。

Objective To increase the yield of bispecific nano-antibody carcinoembroynic antigen(CEA)/CD16 by fusion protein expression system,and purify and identify the expressed product. Methods CEA/CD16 gene was cloned into vector p GEX4 T1,and the constructed recombinant plasmid pGEX4 T1-CEA/CD16 was transformed to E.coli BL21(DE3) and induced with IPTG. The expressed fusion protein GST-CEA/CD16 was purified by GST affinity chromatography,digested with TEV,further purified by Ni-NTA and Q-Sepharose FF chromatography and determined for activity.Results Restriction analysis and sequencing proved that recombinant plasmid pGEX4 T1-CEA/CD16 was constructed correctly. The expressed fusion protein,with a relative molecular mass of about 58 000,mainly existed in a soluble form in supernatant of lysed bacteria. The yield of fusion protein after purification was 5 mg/L. The fusion protein mediated the NK cells to kill CEA positive cancer cells in a dose-dependent pattern. Conclusion GST-CEA/CD16 fusion protein was expressed successfully,which showed high biological activity. It indicated that GST fusion expression was feasible to bispecific antibody.