肝癌抗原肽（EPVTKAEML）及人HSP70融合基因构建及原核表达.docVIP

肝癌抗原肽（EPVTKAEML）及人HSP70融合基因构建及原核表达 　 作者：曲萍, 马加海, 黄勇, 刘利兵, 铁茹, 刘芳娥, 黄小军 【摘要】 　　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（EPVTKAEML）与人HSP70的融合基因。 【关键词】 肝肿瘤/免疫学 抗原 肿瘤/遗传学 表位 热休克蛋白类70/遗传学 基因表达 　　[Abstract] 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 Mr 72 000 fusion protein was expressed. CONCLUSION: The fusion gene of EPVTKAEMLHSP70 has been successfully constructed and expressed in E.coli BL21(DE3). 　　[Keywords]hepatoma/immunology; antigen; neoplasm/genetics; epitope; heatshock protein 70/genetics; gene expression 　　原发性肝癌是严重危害我国人民身体健康的恶性疾病之一。近几十年来, 尽管诊疗手段有了很大的进步, 但肝癌的预后并没有得到显著改善。生物治疗是肝癌治疗的重要手段之一, 尤其可能在复发转移的控制中发挥重要作用。我们应用弱酸洗脱质谱法首次从肝癌细胞系中成功分离了MAGE1编码的由HLAB7提呈的肝癌抗原肽EPVTKAEML, 将其负载DC细胞, 在体外及裸鼠体内均可诱导抗肝癌特异性免疫反应[1, 2]。为加强EPVTKAEML的免疫原性, 选择具有诱导抗肿瘤免疫作用的HSP70为载体, 制备了EPVTKAEML与HSP70的融合蛋白, 为进一步观察融合蛋白能否诱导