摘要
CD3单抗通过多种途径有效地影响机体的免疫状态，在临床应用中具有极大的潜力。为克服鼠源单抗用于临床的局限性，拟采用抗体工程技术研制抗人CD3改形单链抗体。首先，将鼠源CD3单抗OKT3轻重链CDRs分别移植到人源抗体LS1轻链和Nd重链的框架中，经计算机模拟其空间构象，进行残基替换，确定CD3改形VL、VH_氨基酸序列，化学合成改形VL、VH_基因，将其分别插入至载体pROH80中，构建成抗人CD3改形单链抗体（scFv）基因。再将scFv基因克隆至构建的表达载体pALM中诱导表达，产物主要为包涵体。对包涵体进行变性和复性，用IMAC法纯化，FACS法测定CD3改形scFv与抗原的结合活性，显示其竞争抑制率为18%。
Monoclonal_antibody_(McAb)_against human CD3 can adjust human body's immune statement in various ways, so that its clinical potential is highly regarded. In order to overcome the immunogenecity related to the murine McAb, this research effort was focused on constructing a reshaping single-chain antibody(scFv) against human CD3 employing antibody engineering. First, the CDRs of the murine McAb against human CD3 OKT3 was transplanted into the light-chain framework regions (FRs) of human McAb LS1 and the heavy-chain FRs of human McAb Nd respectively, spatial conformation was predicted by computer analysis. Then some particular residues were replaced in FRs basing on the result of conformational prediction to draw out the amino acid sequences of the reshaped VL and VH_. The genes were chemically synthesize and inserted into an expression vector pROH80 to construct the reshaping scFv. Inducing the expression of reshaping scFv, the products are mainly as inclusion bodies. The reshaping scFv was expressed in another vector pALM. The inclusion bodies were denatured and then renatured by gel filtration. The renatured products were purified by immobilized metal affinity chromatograph (IMAC). Finally, the antigen-binding activily of the reshaping scFv against human CD3 was testified by the Compelitire in hibilory fluorescenceactivated cell sorting (FACS). The competitive inhibition rate is 18%.
基金
国家863计划资助!(编号:102-09-04-01)
关键词
CD3
改形抗体
表达载体
构建
reshaping scFv
expression vector
construction
expression
CD3
