转铁蛋白受体单链抗体与链亲和素融合基因的克隆及其在大肠杆菌中的可溶性表达

目的:转铁蛋白受体特异性富含于血脑屏障和肿瘤细胞的表面,是当前中枢神经系统疾病和肿瘤治疗中定向转运的重要靶标。拟获得在大肠杆菌中能高效可溶表达的转铁蛋白受体单链抗体与链亲和素(SA)的重组融合蛋白。方法:根据GenBank数据库报道的SA的核苷酸序列分段合成基因,连接后经PCR获得完整的基因片段,插入pGEM-T载体中测序。将序列正确的SA基因与大鼠转铁蛋白受体单链抗体基因ox26-scFv分别插入原核表达载体pTIG-Trx中,构建重组表达克隆pTIG-Trx/scFv-SA,并在大肠杆菌中诱导表达。ELISA检测融合蛋白的生物学活性。结果:对pGEM-T/SA克隆的测序结果显示,合成的SA基因与文献报道相符。重组融合蛋白在大肠杆菌中获得了可溶性表达,约占菌体上清总蛋白量的30%;ELISA结果表明该融合蛋白具备与转铁蛋白受体和生物素的结合的双重活性。结论:有活性的重组融合蛋白的获得为构建一个通用性的以转铁蛋白受体介导的血脑屏障和肿瘤转运靶向载体打下了基础。

Cloning and Soluble Expression in Escherichia coli of Fusion Gene of Anti-Transferrin Receptor scFv and Streptavidin

Objective: The transferrin receptors are specially enriched on the surfaces of the blood-brain barrier and tumor cells, which are the important target in the central nervous system disease and tumor therapy. To obtain the anti-transferrin receptor scFv and streptavidin(SA) recombinant fusion proteins which were high-efficiently and solubly expressed in E.coli. Methods: The streptavidin gene sequence was synthesized divided into 15 complement segments according to the information of GenBank database, ligated and amplified by PCR. The PCR products were inserted into the pGEM-T vectors and sequenced. The accurate full-length SA gene and the rat single-chain antibody gene(ox26-scFv) to transferrin receptor were inserted into the prokaryotic expression vector pTIG-Trx separately to construct the recombinant expression clones pTIG-Trx/scFv-SA which were expressed in E.coli BL21(DE3). Then the biological activity of the fusion proteins was detected by ELISA. Results: The sequence analysis of pGEM-T/SA clone showed the synthesized gene was coincident with streptavidin gene. The ox26-scFv-SA fusion gene was expressed in E.coli BL21(DE3). The soluble products attained 30% of the total cytoplasm suspension proteins. ELISA showed the fusion proteins could bind to transferrin receptors on the surfaces of rat GH3 cell and the biotins. Conclusion: The recombinant fusion protein with biological activity might be used as a universal blood-brain barrier or tumor cells transport vector mediated by transferrin receptor.