论文部分内容阅读
目的:构建和表达抗血管内皮细胞生长因子受体2(VEGFR2)/抗CD3双特异单链抗体(bscVEGFR2×CD3),及亲和活性测定。方法:设计抗VEGFR2/抗CD3双特异单链抗体基因序列,由公司合成并亚克隆入真核表达载体pcDNA3.1(+)中,脂质体法转染中国仓鼠卵巢细胞(CHO),筛选高效分泌表达bscVEGFR2×CD3的克隆株。表达产物经Ni-NTA柱纯化,120 g/L SDS-PAGE电泳及Western blot鉴定。应用流式细胞术(FCM)检测生物学活性。结果:bscVEGFR2×CD3重组表达载体测序证实序列正确。bscVEGFR2×CD3能够在CHO细胞中进行分泌表达,筛选出6株高表达克隆株。120 g/L SDS-PAGE显示相对分子质量(Mr)约56 000有1条带,与预期相符,Western blot证明anti-His抗体能与这条蛋白条带发生特异性结合。FCM检测bscVEGFR2×CD3能与CD3+jurkat细胞和VEGFR2+A375细胞特异性结合。结论:成功构建和表达抗VEGFR2/抗CD3双特异单链抗体,该抗体具有与VEGFR2、CD3特异性结合的免疫学活性。
OBJECTIVE: To construct and express anti-VEGFR2 / bscVEGFR2 × CD3 and to determine the affinity of VEGFR2 / CD3. Methods: The anti-VEGFR2 / anti-CD3 bispecific single-chain antibody gene sequence was designed and synthesized by the company and subcloned into the eukaryotic expression vector pcDNA3.1 (+), transfected into Chinese hamster ovary cells (CHO) Clone Expressing bscVEGFR2 × CD3 Efficiently. The expressed product was purified by Ni-NTA column and identified by 120 g / L SDS-PAGE electrophoresis and Western blot. The biological activity was detected by flow cytometry (FCM). Results: Sequencing was confirmed by sequencing of bscVEGFR2 × CD3 recombinant vector. bscVEGFR2 × CD3 was secreted in CHO cells, and 6 highly expressed clones were screened. 120 g / L SDS-PAGE showed that a relative molecular mass (Mr) of about 56 000 had 1 band, in line with the expectation. Western blot showed that the anti-His antibody specifically bound to this protein band. FCM showed that bscVEGFR2 × CD3 could specifically bind to CD3 + jurkat cells and VEGFR2 + A375 cells. Conclusion: The anti-VEGFR2 / anti-CD3 bispecific single chain antibody was successfully constructed and expressed. The antibody has the immunological activity of binding specifically to VEGFR2 and CD3.