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目的:构建及原核表达肝癌抗原肽(EPVTKAEML)与人热休克蛋白70(HSP70)的融合基因。方法:采用加端PCR方法,将EPVTKAEML的基因序列融合到人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(+)/EPVTKAEML-HSP70。经限制性内切酶BamHI、XhoI双酶切鉴定及序列测定后,转化E.coliBL21(DE3),经IPTG诱导表达融合蛋白,SDS-PAGE检测表达结果。结果:应用加端PCR方法扩增出约2.0kb的目的片段,序列测定结果证实,EPVTKAEML的基因序列成功地融合到人HSP70基因的3′端。经BamHI、XhoI酶切鉴定证实,融合基因成功地克隆到原核表达载体pET-28a(+)上;转入重组质粒的E.coliBL21(DE3)经IPTG诱导后,SDS-PAGE分析发现在相对分子质量(Mr)约72000处有表达量明显增多的蛋白条带。结论:成功地构建并表达了肝癌抗原肽(EPVT-KAEML)与人HSP70的融合基因。
Objective: To construct and prokaryotic express fusion gene of hepatocellular carcinoma antigen peptide (EPVTKAEML) and human heat shock protein 70 (HSP70). Methods: The gene sequence of EPVTKAEML was fused to the 3 ’end of human HSP70 gene by polymerase chain reaction (PCR). The fusion gene was cloned into prokaryotic expression vector pET-28a (+) to construct recombinant plasmid pET-28a (+) / EPVTKAEML -HSP70. After restriction endonuclease digestion with restriction enzyme BamHI and XhoI and sequence determination, the recombinant plasmid was transformed into E.coli BL21 (DE3). The fusion protein was induced by IPTG and the expression was detected by SDS-PAGE. Results: The target fragment of about 2.0 kb was amplified by PCR. The sequencing results confirmed that the gene sequence of EPVTKAEML was successfully fused to the 3 ’end of human HSP70 gene. After digestion with BamHI and XhoI, the fusion gene was successfully cloned into prokaryotic expression vector pET-28a (+). After induced by IPTG, the recombinant plasmid was transformed into E.coli BL21 (DE3) There are about 72,000 protein (Mr) protein bands with significantly increased expression. Conclusion: The fusion gene of hepatocellular carcinoma antigen peptide (EPVT-KAEML) and human HSP70 was successfully constructed and expressed.