原核表达系统pET28a(＋)中抗CD20微抗体的构建和表达

Construction and Expression of Anti-CD20 Minibody in pET28a(+) of Prokaryotic Expression System

目的:改造Rituximab,构建包含VL、VH和CH3的抗CD20微抗体minibody,并探索诱导其可溶性表达的最佳方法.方法:首先通过overlap PCR将Rituximab的VL和VH通过Linker连接在一起,成为单链抗体ScFv,再将ScFv和人源IgG1 CH3通过人源IgG1铰链区连成minibody的cDNA.将其克隆至表达载体pET28a(+)中,并在大肠杆菌中用IPTG诱导表达.结果:SDS-PAGE和Western blot结果表明,抗CD20的微抗体基因表达产物分子量约41.88 kDa,在20℃、1.0 mmol/L IPTG诱导24 h时minibody的可溶性表达量最大,同时还有包涵体形式的蛋白.可溶性抗体蛋白通过镍柱纯化,纯度达到90.25%,包涵体经过变、复性获得了高纯度的抗体蛋白.重组蛋白minibody可特异性地被兔抗人IgG1单克隆抗体识别,并且可特异性结合靶抗原CD20.结论:抗CD20微抗体minibody基因在此原核表达系统中成功表达,为其生物学功能和更好地针对B淋巴系统恶性肿瘤的靶向治疗奠定了基础.

Objective The research aimed to construct anti-CD20 minibody containing VL,VH and CH3,and to explore the best inducing condition of its soluble protein expression. Method The recombinant minibody cDNA was produced by overlap PCR. A short peptide linker was used to join VL and VH. The human IgG1 hinge region was used to join VH and CH3. The recombinant minibody gene was subcloned into the expression vector of pET28a and expressed in E. coli BL21. Result SDS-PAGE and Western blot analysis showed that the recombinant protein was about 41.88 kDa and the optimized soluble protein expression condition of minibody was 1.0 mmol/L IPTG for 24 h at 20~C ,the inclusion bodies were found in the precipitation after sonication. The soluble protein minibody was further purified Ni-NTA affinity chromatography, the yield up to 90. 25% , the highly purity recombinant protein was obtained after a series of steps including inclusion bodies cell lysis, denaturation, purfication and renaturation. Western blot proved that the recombinant protein anti-CD20 minibody had immunological reactive ability against rabbit anti-human IgG, and can specific binding CD20 antigen. The successful expressed of minibody in the prokaryotic expression system was helpful for the future study of its biological function and target therapy to the B lymphoid leukemia and B lymphoma.