论文部分内容阅读
目的构建MAGE-n159-167(QLVFGIEVV)表位肽与人热休克蛋白70(HSP70)的融合基因并表达纯化。方法应用PCR方法,将QLVFGIEVV的cDNA序列融合到人HSP70基因的3’端,将融合基因克隆入原核表达载体pET-28a(+)中,构建重组质粒pET-28a(+)QLVFGIEVV-HSP70;采用双酶切(Sac Ⅰ、Hind Ⅲ)及PCR鉴定后测序;转化大肠杆菌E.coli BL21(DE3),用IPTG诱导表达,Ni Sepharose6FF亲和填料进行分离纯化,SDS-PAGE检测表达及纯化结果。结果经PCR扩增成功获得约2.0kb的目的片段,重组体经Sac Ⅰ、Hind Ⅲ双酶切分析及PCR结果与预期结果一致,测序正确。转入重组质粒的E.coli BL21(DE3)经IPTG诱导、SDS-PAGE分析得到71kD左右的目的蛋白条带。用Ni Sepharose6FF亲和填料分离纯化,获得了纯化的融合蛋白。结论成功构建原核表达载体pET-28a(+)QLVFGIEVV-HSP70,并获得纯化的融合蛋白,为基于MAGE-n的肿瘤疫苗研制提供良好的抗原奠定了实验基础。
Objective To construct and express the fusion gene of MAGE-n159-167 (QLVFGIEVV) epitope peptide and human heat shock protein 70 (HSP70). Methods The cDNA sequence of QLVFGIEVV was fused to the 3 ’end of human HSP70 gene by PCR. The fusion gene was cloned into prokaryotic expression vector pET-28a (+) to construct recombinant plasmid pET-28a (+) QLVFGIEVV-HSP70. The recombinant plasmid was digested by Sac Ⅰ and Hind Ⅲ and sequenced. The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG. The recombinant protein was purified by Ni Sepharose 6FF affinity chromatography and expressed by SDS-PAGE. Results The target fragment of about 2.0 kb was successfully obtained by PCR amplification. The recombinant was digested by Sac Ⅰ and Hind Ⅲ and the PCR result was consistent with the expected result. The sequencing was correct. The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG. SDS-PAGE analysis yielded a band of about 71kD. Purified with Ni Sepharose 6FF affinity packing to obtain the purified fusion protein. Conclusion The prokaryotic expression vector pET-28a (+) QLVFGIEVV-HSP70 was successfully constructed and the purified fusion protein was obtained, which provided the experimental basis for the development of MAGE-n-based tumor vaccine.