全人源药用抗EGFR ScFv（HL-L-L）的构建、表达及抗肿瘤活性分析

摘要

Objective To construct,express and analysis the antitumor activity of anti-EGFR ScFv(HL-L-L),and analyze bioinformatics and characterize correlation spectroscopy.Methods EGFR ScFv(HL-L-L)gene was obtained by OVER LAPING technology and construction of prokaryotic expression vector pET-28a-c(+)-VH-L-L.After induced expression,target protein HL-L-L was purified by Ni column,and its tertiary structures was predicted with SWISS - MODEL;size distribution and zeta potential distribution of the target protein HL-L-L were characterized by NanoZSP;the inhibition effects of HL-L-L on breast cancer cell proliferation were studied with the method of pharmacology.Results Prokaryotic expression vector pET-28a-c(+)-VH-L-L was constructed successfully,high purity of protein HL-L-L was obtained,and successfully predicted its three-dimensional structure;the size was about 489.7 nm,and the Zeta potential was 38.1 ,negatively charged.It had a certain inhibition effect on MCF # 7 proliferation,compared with blank group,and the difference was significant(P<0.01 ).Conclusion This study successfully express all anthropogenic medicinal anti-EGFR ScFv(HL-L-L),which has significant inhibiting effect on breast cancer cell proliferation.It provides the foun dation for further development of small molecules of anti-EGFR antibody drug.%目的：对抗EGFR单克隆抗体重链可变区（VH）和轻链全长（L）经柔性肽链（linker）连接而成的单链抗体（VH-L-L）进行构建、表达及其抗肿瘤活性的分析，并对其进行生物信息学分析和相关谱学表征。方法通过OVER LAPING技术得到EGFR ScFv （HL-L-L）基因，然后构建原核表达载体pET-28a-c（＋）-VH-L-L，经诱导表达、Ni柱纯化后得到目的蛋白HL-L-L，并对其利用SWISS-MODEL进行三级结构预测；通过紫外扫描分光光度计，进行吸收波长的扫描；用NanoZSP表征目的蛋白VH-L-L的粒度和电位；随后利用药理学方法，研究HL-L-L对乳腺癌细胞的增殖抑制情况。结果成功构建了原核表达载体pET-28a-c（＋）-VH-L-L；获得了较高纯度的VH-L-L蛋白，并成功预测了其三维结构；其粒度约为489.7 nm，Zeta电势为-38.1，带负电；HL-L-L对MCF-7具有一定的增殖抑制作用，与空白组相比差异有统计学意义（P＜0.01）。结论成功表达了全人源药用抗EGFR ScFv（HL-L-L），其对乳腺癌细胞的增殖抑制作用显著，为下一步研制抗EGFR的小分子抗体药物提供基础。