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目的:设计原核表达小鼠钙网蛋白(Calreticulin,CRT)与程序性死亡分子1(Programmed Death-1,PD1)的融合蛋白,用于肿瘤靶向治疗。方法:应用RT-PCR技术扩增小鼠CRT的N、P区基因及PD1胞外区cDNA序列,用一个Linker将两者连接起来,克隆至表达载体pET28a(+)上。将重组表达质粒pET28a(+)-CRT-SPD1转化入大肠杆菌BL21(DE3)内,IPTG诱导表达;用Ni柱亲和层析纯化蛋白,并进行SDS-PAGE、Western blot鉴定。结果:所得产物经SDS-PAGE检测后,在45 kDa处显示特异条带,最后经Western Blotting鉴定,所纯化蛋白能被CRT抗体和PD1抗体抗体识别。结论:获得较纯的融合蛋白,所构建的载体可为进一步研究两者的功能和免疫学特性及肿瘤的免疫治疗奠定了基础。
Objective: To design a fusion protein with prokaryotic expression of mouse calreticulin (CRT) and programmed death-1 (PD1) for tumor targeted therapy. Methods: The cDNA of N and P region and PD1 extracellular region of mouse CRT were amplified by RT-PCR. The two sequences were ligated by a Linker and cloned into the expression vector pET28a (+). The recombinant plasmid pET28a (+) - CRT-SPD1 was transformed into E. coli BL21 (DE3) and induced by IPTG. The protein was purified by Ni affinity chromatography and identified by SDS-PAGE and Western blot. Results: The product was detected by SDS-PAGE at 45 kDa showed a specific band, and finally identified by Western Blotting, the purified protein can be antibodies to antibodies and antibodies to PD1 PD1. Conclusion: Obtaining pure fusion protein, the constructed vector can lay a foundation for the further study of both functional and immunological characteristics and tumor immunotherapy.