抗人血管内皮生长因子单链抗体基因的构建、表达及活性鉴定

Construction and expression of the variable gene encoding a single-chain antibody against human VEGF

鼠单克隆抗体E11能与人血管内皮生长因子(VEGF)特异结合,已用于临床检测恶性肿瘤细胞VEGF的表达,并初步证明其体内抑瘤活性。为便于大规模生产,特进行基因工程改造,构建小分子的单链抗体(scFv)。首先通过逆转录及多聚酶链式反应(PCR),分离并克隆E11的可变区基因。经测序表明,E11轻链可变区(VL)基因全长333bp,编码111个氨基酸,归属小鼠轻链可变区基因第Ⅲ亚组。重链可变区VH基因全长369bp,编码123个氨基酸,归属小鼠重链可变区基因Ⅱ(A)亚组。然后用一编码亲水性多肽接头的DNA片断将E11单抗轻、重链可变区基因连接,构建表达质粒pET-15YV,在大肠杆菌BL21(DE3)中进行表达。表达产物(包含体)经变性及复性后,用免疫组化法检测该单链抗体结合抗原(颊癌)能力。对颊癌组织检测的结果表明,基因工程抗体scFv与亲代抗体一样,具有较高的组织特异性。本研究获得的抗人VEGF单链抗体具有潜在的临床价值,为肿瘤放射免疫显像及以血管为靶标的抗血管生成治疗奠定了基础。

A rodent hybridoma, which reacts specifically with human VEGF, has been used to screen the VEGF expression of tumors in clinical as well as proved to inhibit the growth of tumors in vivo. For large scale production by engineering methods and widening application of this monoclonal antibody (Mab) in both diagnosis and treatment of human cancer, the variable gene of E11 was isolated to construct single-chain antibody (scFv). Firstly the total RNA of the hybridoma was prepared and used as a template for cDNA synthesis by reverse transcription. Polymerase chain reaction(RT-PCR) were used to amplify the immunoglobulin variable region gene (VH)VL). Sequence analysis revealed that the full length of VH is 369bp, and VL, 333bp. According to Kabat's classification, VH and VL are member of mouse Ig variable gene heavy chain subgroup Ⅱ (A) and light chain subgroup Ⅲ , respectively. The cloned VH and VL were linked by a flexible peptide bridging sequence (GGGGS)3 in the VH-linker-VL orientation. The constructed scFv gene was inserted in pET-15s vector and expressed in E. coli BL21(DE3). The scFv was highly produced in the form of inclusion body. Binding ability of the scFv was determined by immunochemical methods after the inclusion body was denatured and refolded. Result of immunochemical method showed that the scFv retained the simi- lar binding ability specificity as its parent monoclonal antibody. The scFv is potential in both diagnostic and therapeutic application in such fields as radioimmnnoimagmg (R Ⅱ ) and tumor vascular-targeted anti-angiogenesis therapies.