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目的:表达人肿瘤蛋白D52(human tumor proteinD52,hTPD52)的重组融合蛋白,并对该蛋白的生物学特性进行鉴定。方法:RT-PCR扩增乳腺癌MCF-7细胞中的hTPD52基因并克隆于pMD18-T载体中,测序鉴定序列正确后,分别插入pET-32a、pGEX-4T-2和pET-28a3种表达载体中。重组质粒经酶切鉴定、序列分析正确后转化大肠杆菌BL21(DE3),选择最佳表达载体和表达条件进行大量表达,表达产物经His柱亲和纯化和超滤浓缩后,应用Dot blot、Western blot鉴定纯化蛋白的特性。结果:正确扩增出了hTPD52基因,鉴定其为isoform3型。用pET-32a-hTPD52重组表达载体能够大量表达可溶性的hTPD52-Trx融合蛋白,Dot blot显示表达的融合蛋白能和抗hTPD52的抗体结合,SDS-PAGE和Western blot显示表达的融合蛋白分子质量约为44ku,去除Trx后仍能够与特异性抗体结合。结论:成功制备hTPD52重组融合蛋白,并证明原核表达的hTPD52保留了原有的抗原结合活性。
Objective: To express the recombinant fusion protein of human tumor protein D52 (hTPD52) and identify the biological characteristics of the protein. Methods: The hTPD52 gene was amplified by RT-PCR from breast cancer MCF-7 cells and cloned into pMD18-T vector. The sequence of the hTPD52 gene was confirmed by sequencing and inserted into expression vector pET-32a, pGEX-4T-2 and pET-28a in. The recombinant plasmid was identified by restriction enzyme digestion and sequenced. The recombinant plasmid was transformed into E.coli BL21 (DE3), and the recombinant plasmid was selected for expression under the optimal conditions. The recombinant protein was purified by affinity chromatography on His column and concentrated by ultrafiltration. blot to identify purified protein properties. Results: The hTPD52 gene was correctly amplified and identified as isoform3. The soluble hTPD52-Trx fusion protein was expressed in large quantities by the recombinant expression vector pET-32a-hTPD52. Dot blot showed that the expressed fusion protein could bind to the anti-hTPD52 antibody. The molecular weight of the expressed fusion protein was about 44ku, after Trx removal can still be combined with specific antibodies. Conclusion: The hTPD52 recombinant fusion protein was successfully prepared and proved that the prokaryotic expression of hTPD52 retained the original antigen binding activity.