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由于免疫原性,鼠源性单克隆抗体在人体疾病治疗方面受到限制,运用基因重组技术,将抗体轻链、重链可变区基因连接,构建单链Fv(ScFv)基因.表达制备ScFv片段,由于其分子小,免疫原性弱,有潜在应用前景.本实验将获得的抗人肺癌单克隆抗体轻重链可变区基因,通过Linker基因序列连接并克隆至pIg20表达载体,表达制备抗人肺癌scFv融合蛋白.现报道如下.l 材料与方法1.1 抗人肺癌单克隆抗体3D_3(McAb3D_3)重链可变区基因(VH)克隆至表达载体pIg20,以SmaⅠ,XbaⅠ分别双酶切VH及pIg20质粒,以粘末端方式,将SmaⅠ-VH-Xba片段连接到pIg20载体,连接产物电穿孔法转染BL21菌,筛选鉴定.阳性子命名为pIgVH.1.2 将McAb3D_3轻链可变区基因(VL)克隆至pIgVH,以BgLⅡ、NcoⅠ分别消化中pIgVH及VL,以粘
Due to the immunogenicity, mouse-derived monoclonal antibodies are limited in the treatment of human diseases. Genetic recombination technology is used to link the genes of the variable regions of the light and heavy chains of antibodies to construct a single-chain Fv (ScFv) gene. ScFv fragments are expressed and expressed. Due to its small molecule and weak immunogenicity, it has potential application prospects. In this experiment, the light and heavy chain variable region genes of human anti-human lung cancer monoclonal antibodies were obtained and linked by Linker gene sequences and cloned into pIg20 expression vector for expression and anti-human preparation. The scFv fusion protein of lung cancer is reported as follows. l Materials and methods 1.1 The anti-human lung cancer monoclonal antibody 3D_3 (McAb3D_3) heavy chain variable region gene (VH) was cloned into the expression vector pIg20, which was double digested with SmaI, XbaI and pIg20 respectively. Plasmid, in the form of sticky ends, the SmaI-VH-Xba fragment was ligated into the pIg20 vector and the product was electroporated into the BL21 strain and screened for identification. The positive clone was designated as pIgVH.1.2. The McAb3D_3 light chain variable region gene (VL) After cloning into pIgVH, pIgVH and VL were digested with BgLII and NcoI, respectively.