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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。
OBJECTIVE: To identify and purify the expression product by using NY-ESO-1 human CT antigen expressed in Escherichia coli, and to lay the foundation for its future research on tumor vaccine. Methods: NY-ESO-1 gene was obtained by whole gene splicing. The recombinant expression vector NY-ESO-1-pET28a (+) was constructed and expressed in E.coli BL21 (DE3) by IPTG. Identification, affinity purification by Ni column to obtain purified protein. RESULTS AND CONCLUSION: The full-length NY-ESO-1 spliced gene was verified by sequencing to be completely consistent with the human NY-ESO-1 gene in GenBank. The constructed prokaryotic expression vector NY-ESO-1-pET28a NY-ESO-1 protein was expressed. The purified product was purified by Ni-affinity column. The purified protein was verified by mouse anti-human monoclonal antibody. The result showed that the protein was positive. The successful use of prokaryotic expression system to achieve the CT antigen NY-ESO-1 soluble expression.