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目的鼠/人源化抗人肝癌单链抗体(m/hscFv_(25))融合人突变型 TNFα(mTNFα)原核表达载体的构建及表达.方法用 Oligo 软件分析并设计人 mTNFα的5′及3′端引物,化学合成并用 PCR 方法扩增 mTNFα,经限制性内切酶酶切后,连接在原核表达载体 pGEX4T-1中的 m/hscFv_(25)之后,构建成pGEX4T-1/m/hscFv_(25)-mTNFα融合蛋白表达载体,转化大肠杆菌 JM109后,通过 IPTG 诱导进行目的蛋白的初步表达.结果经 DNA 电泳分析表明,mTNFα基因全长约460 bp,并通过酶切鉴定证明成功构建了抗人肝癌单链抗体融合 mTNFα的原核表达载体,融合蛋白产物经 SDS-PAGE 显示,相对分子质量(Mr)为68 000的蛋白谱带,与预期设计完全一致,光密度扫描结果表明,GST-mscFv_(25)-mTNFα和 GST-hscFv_(25)-mTNFα融合蛋白分别占菌体总蛋白的15%和12%,表达产物主要以不溶性包涵体形式存在.结论成功构建并表达了鼠/人源化抗人肝癌单链抗体融合人mTNFα的双功能抗体基因,并在原核细胞中获得较高水平的表达,为进一步研究 HCC 的治疗奠定了基础.
Objective To construct and express prokaryotic expression vector of murine / humanized anti-human hepatocellular carcinoma single chain antibody (m / hscFv_ (25)) fused with human mutant TNFα (mTNFα) .Methods Oligo software was used to analyze and design 5 ’and 3 MTNFα was amplified by PCR and cloned into pGEX4T-1 / m / h scFv_ (25) by restriction endonuclease digestion and ligated with m / hscFv_ (25) in prokaryotic expression vector pGEX4T- (25) -mTNFα fusion protein was constructed and transformed into E.coli JM109.The expression of target protein was induced by IPTG.Results The full length of mTNFα gene was about 460 bp by DNA electrophoresis analysis, and confirmed by restriction enzyme digestion that it was successfully constructed The prokaryotic expression vector of mTNFα fused with single chain antibody against human hepatocellular carcinoma was constructed. The SDS-PAGE showed that the relative molecular mass (Mr) of the protein was 68 000, which was completely consistent with the expected design. The results of optical density scanning showed that GST- The mscFv_ (25) -mTNFα and GST-hscFv_ (25) -mTNFα fusion proteins accounted for 15% and 12% of the total bacterial proteins, respectively, and the expressed products mainly existed as insoluble inclusion bodies.CONCLUSIONS Mouse / human Anti-human hepatoma single chain antibody fusion Bifunctional antibody genes mTNFα and obtain higher levels of expression in prokaryotic cells, which laid a foundation for further study of the treatment of HCC.