胃癌MG-7抗原模拟表位与HSP70融合基因的构建及表达

Construction and expression of gastric cancer MG7 mimic epitope fused to heat shock protein 70

目的构建和表达胃癌 MG7模拟表位与人热休克蛋白70(HSP70)的融合基因.方法采用加端 PCR 的方法,将 MG7模拟表位(GC23)的编码序列融合到 HSP 基因的3′端;PCR 获得的融合基因片段经 T-A 克隆法克隆到 pUCm-T 载体上,并进行 DNA 测序;将融合基因片段从 pUCm-T 载体上酶切后,亚克隆到表达载体 PET-21a(+)上;将重组质粒 PET-21a(+)/GC23-hsp 转化大肠杆菌,经IPTG 诱导后,收集细菌,菌体裂解后进行 SDS-PAGE 及Western blot 检测.结果应用 PCR 方法扩增出约2.0kb 的目的片段,序列测定结果证实,模拟表位 GC23序列成功地融合到 HSP70基因的3′端,GC23及 HSP70基因序列均正确;经 EcoR I+Xho I 酶切及 PCR 鉴定证实,融合基因成功地克隆到原核表达载体 PET-21a(+)上;转入重组质粒的大肠杆菌经 IPTG 诱导后,进行SDS-PAGE 发现在 M_r 为72000处有表达量明显增多的蛋白条带,Western blot 证实,M_r72000处的条带为目的条带.结论成功构建并表达了 MG7模拟表位与 HSP70的融合基因.

AIM To construct and express in E.coli a fusion gene of gastric cancer MG7 mimic epitope with heat shock protein 70 (hsp70). METHODS A cDNA fragment encoding MG7 mimic epitope (GC23) was added to 3(?) terminus of hsp70 gene by PCR ampUfication.The PGR products were cloned into pUCm-T vector and were sequenced.The fusion gane was subcloned into vector PET-21a (+) and expressed in E. coli.SOS-PAGE and Western blot were employed to identify the expression of the fusion gene. RESULTS A fragment about 2.0kb was amplified by the PCR.Sequence analysis revealed that the sequence of GC23 was connected successfully to 3(?) terminus of hsp70. The fusion gene was cloned into PET-21a(+) identified by enzyme digestion and PCR.SOS-PAGE and Western blot showed that a M_r72 000 fusion protein was expressed that could be recognized by anti-hap70 antibody. CONCLUSION The fusion gene of GC23/hap70 has been successfully constructed and expressed in E.coli.