抗人整合素αvβ3-组织因子融合蛋白基因的构建和表达

Construction and Expression of a Fusion Protein of a Single Chain Antibody to Human Integrin αvβ3 and Tissue Factor

目的:利用基因工程技术构建和表达抗人整合素αvβ3单链抗体(scFv)与人可溶性组织因子(sTF)的融合蛋白scFv-sTF。方法:用重叠延伸PCR技术扩增scFv基因,同时用组装PCR方法人工合成sTF基因,然后以酶切连接方式融合2个基因,并克隆至原核表达载体pQE80L中,构建表达质粒pQE80L-scFv-sTF,以重组子转化大肠杆菌M15诱导目的基因表达。结果:SDS-PAGE分析显示工程菌可以表达相对分子质量约55×103的融合蛋白scFv-sTF,Western印迹分析证实表达的目的蛋白具有6×His标签;经低剂量IPTG诱导和较低温度培养,scFv-sTF获得了可溶性表达;纯化回收目的蛋白,ELISA试验证实,该重组抗体分子具有良好的抗原结合活性。结论:构建并表达了抗人整合素αvβ3的单链抗体融合蛋白scFv-sTF,并验证了其抗原结合活性,初步证明它可以与抗原特异性结合,为进一步研究其抗肿瘤作用打下了基础。

Objective： To construct and express a fusion protein of a single chain antibody（scFv） to human inte-grin αvβ3 and soluble human tissue factor（sTF）. Methods： The scFv gene was amplified by overlapping extension PCR and the sTF gene was synthesized by assembly PCR technique, and they were joined together as a fusion gene and were cloned into the prokaryotic expression vector pQE80L to construct the expression plasmid pQE80L-scFv-sTF, which was expressed in E.coli M15. Results： SDS-PAGE analysis showed that the expressed recombined protein had a molecular weight of 55 kD. Western blot analysis confirmed that the protein was tagged with 6×His. The scFv-sTF protein was expressed as soluble protein under the condition of low concentration IPTG induction at low temperature. The reconbinant protein was purified and ELISA suggested that the recombinant fusion protein could bind to the human integrin αvβ3. Conclusion： The fusion protein scFv-sTF have been expressed, which lays the foundation for the analasis of its anti-tumor activities.