肝癌抗原肽(EPVTKAEML)与人HSP70融合基因的构建及原核表达

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BamH I、Xho I双酶切鉴定及序列测定后,转化E.coli BL21(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2.0 kb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BamH I、Xho I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.coli BL21(DE3)经IPTG诱导后,SDS-PAGE分析发现在相对分子质量(Mr)约72 000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(EPVTKAEML)与人HSP70的融合基因.

Construction and expression of a fusion gene of human heptoma peptide with human heat shock protein 70

AIM:To construct and express a fusion gene of human heptoma peptide (EPVTKAEML) with human heat shock protein 70 (HSP70). METHODS: A cDNA fragment encoding EPVTKAEML was added to 3 terminus of human HSP70 gene by PCR amplification. The PCR products of fusion gene were cloned into pET-28a(+)vector. The recombinant plasmid pET-28a(+)/EPVTKAEML-HSP70 was identified by enzyme digestion analysis and sequencing, and then it was transformed into E.coli BL21(DE3) through IPTG induction to express the target protein bearing His tag. RESULTS: A fragment of about 2.0 kb was amplified by PCR. Sequence analysis revealed that the sequence of EPVTKAEML was connected successfully to 3 terminus of human HSP70. Enzyme digestion analysis showed the fusion gene was cloned into pET-28a(+). SDS-PAGE showed that a relative molecular mass 72 000 fusion protein was expressed. CONCLUSION: The fusion gene of EPVTKAEML-HSP70 has been successfully constructed and expressed in E.coli BL21(DE3).