摘要
单链抗体(Single-chain，Fv)是利用基因工程的方法，将免疫球蛋白重链可变区(V_H)和轻链可变区(V_L)用一连接肽(Linker)相连接而成的，具有抗原特异性强、分子量小、组织穿透力强、体内滞留时间短、血中清除快、无Fc段所引起的非特异性反应、C-端能与其它分子相连接等优点，因而在肿瘤临床定位诊断、导向性治疗以及基础理论研究等方面具有十分重要的作用。抗人纤维蛋白单链抗体scFv-8E5的轻、重链可变区基因是从抗人纤维蛋白单克隆细胞系8E5中提取的，该单链抗体能特异性识别人纤维蛋白β链7肽，因而在血栓病的定位及导向溶栓等方面有诱人的应用前景。近来的研究表明，单链抗体若能形成二聚体结构，可以明显提高其抗原结合力及其自身的稳定性。同时，用连接肽连接异源的轻、重链可变区基因，形成的二聚体可以同时识别二种不同的抗原，即具有双特异性，这在临床应用中具有十分重要的价值。本文利用已有的抗人纤维蛋白单链抗体scFv-8E5，利用基因工程的方法，对其连接肽进行了改造，分别用三种连接肽取代原有连接肽以研究这些连接肽对该单链抗体二聚体形成、抗原结合力、稳定性的影响。
方法：以人工合成三种新的连接肽DNA作为引物的一部分，经PCR扩增分别将这三种新连接肽插入V_H与V_L之间，取代原有的连接肽，用SDS-PAGE观察表达结果，用ELISA法检测其抗原结合力及稳定性。
结果：本文新构建的三种质粒pOPE51-8E5-5、pOPE51-8E5-10A、pOPE51-8E5-10B均能在大肠杆菌JM109中表达，它们的表达产量均高于
Single-chain Fv (ScFv) is recombinant antibody fragment consisting of only the variable light chain (V_L) and variable heavy chain (V_H) domains covalently connected to one another by a polypeptide linker. Due to the small size, the scFv shows faster plasma clearance and stronger tissue penetration in vivo, and easier being linked to another effector molecules at the C-terminal. It has binding specificity as its parent antibody does, but has no non -specific binding induced by the Fc fragments. These advantages favour the scFv applications as imaging reagents for tumor localization and targeting delivery of drugs, toxins or radionuclides to the tumor. ScFv-8E5 is a 30KDa single chain Fv with the antigen-binding specificity against a synthetic heptapeptide of the β chain of human fibrin. Therefore, it may be useful in thrombosis imagine and targeting thrombolytic therapy. Investigation data shows that bivalent scFv has better antigen-binding specificity and stability than those of the monomer. It can also be formed by two heterogenic scFvs as bifunctional biabody.
In this studies, the linker of the scFv-8E5 has been modified. The effects of the modifications on (scFv)_2 dimer formation and antigen binding have been carried out.
Methods : The DNA fragments of three linkers were synthesized as parts of the primers. Using PCR techniquence, each linker was inserted between the V_H and V_L to replace the primary linker in pOPE51-8E5. Three
引文
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