CT抗原NY-ESO-1基因克隆、表达及纯化

Molecular cloning,prokaryotic expression and purification of human CT antigen NY-ESO-1

目的:利用大肠杆菌表达人CT抗原NY-ESO-1,对表达产物进行Western印迹鉴定和纯化,为今后利用其进行肿瘤疫苗的研究奠定基础。方法:通过全基因拼接获得NY-ESO-1基因,构建重组表达载体NY-ESO-1-pET28a(+),在大肠杆菌BL21(DE3)中利用IPTG诱导获得表达,利用单克隆抗体进行Western印迹鉴定,通过Ni柱亲和纯化获得纯化蛋白。结果与结论:拼接的NY-ESO-1全基因经测序证明与GenBank中人的NY-ESO-1全基因完全相符;构建的原核表达载体NY-ESO-1-pET28a(+)可稳定地可溶性表达NY-ESO-1蛋白;经Ni柱亲和纯化获得较好的纯化结果;利用鼠抗人单克隆抗体对表达的蛋白进行验证,结果显示阳性。本研究成功利用原核表达系统实现了对CT抗原NY-ESO-1的可溶性表达。

Objectives：To clone,and express the CT antigen NY-ESO-1 in E. coli, to identify the protein by Western blot and purify the protein for further investigation as tumor vaccine. Methods：The full-length gene of NY-ESO-1 was generated by gene splicing method and the recombinant expression vector NY-ESO-1-pET-28a （ ＋ ） was constructed, E, coli BL21 （DE3） bearing the plasmid was induced with IPTG for protein production, Target protein was characterized by We, stern blot with monoclonal antibody and purified by Ni^2＋ affinity chromatography. Results and Conclusion：The full-length gene confirmed by sequence analysis matched human gene NY-ESO-1 in GenBank exactly. The reconstructed vector NY- ESO-1-pET-28 could express target protein stably in the soluble fraction of bacterial extract. NY-ESO-1 protein could be purified by Ni^2＋ affinity chromatography. To confirm the target protein, Western blot was performed with mouse anti-human NY-ESO-1 monoclonal antibody. The result was positive. So human CT antigen NY-ESO-1 could be successfully expressed in prokaryotic expression system.