Zhang M, Yu L, Huang H
Institute of Urology, Peking University, Beijing 100034, China.
Zhonghua Wai Ke Za Zhi. 2001 Oct;39(10):792-5.
To construct and express single chain Fv antibody against human bladder carcinoma, which was expected to have advantages in targeted diagnosis and therapy with the characteristics of lower immunogenicity.
Hybridoma BDI-1 cell, which secreted a monoclonal antibody against human bladder carcinoma, was used to isolate total RNA. By reverse transcription, the cDNA was synthesized and used as templates for amplifying the immunoglobulin heavy-and light-chain variable region genes by polymerase chain reaction (PCR). The amplified DNA was ligated into a sequencing vector pUC19 and sequenced with Sanger's method. The VH and VL genes were inserted into expression vector pFUW80. By inducing, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected by ELISA. The 5 x his-tagged ScFv antibodies were purified on IDA-Ni2+ resin by immobilized metal chelate affinity chromatography (IMAC). The purified ScFv antibodies were analyzed by SDS-PAGE.
A full-length of VH and VL genes was 366 and 324 base pairs respectively. Comparing with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from re-arrangement of VH, Dsp2.2 and JH4; the VL gene was VK subgroup IV and from Vk and Jk4. The ScFv antibodies could inhibit 84% of the antigen binding activity of original McAb BDI-1. The purified ScFv antibodies gave a single major band (Mr-29 000) on SDS-PAGE.
The single chain Fv antibody against human bladder carcinoma was successfully constructed and expressed.
构建并表达抗人膀胱癌单链Fv抗体，期望其具有免疫原性较低的特点，在靶向诊断和治疗方面具有优势。
采用分泌抗人膀胱癌单克隆抗体的杂交瘤BDI-1细胞分离总RNA。通过逆转录合成cDNA，以此为模板用聚合酶链反应（PCR）扩增免疫球蛋白重链和轻链可变区基因。将扩增的DNA连接到测序载体pUC19中，采用桑格法进行测序。将VH和VL基因插入表达载体pFUW80。通过诱导，从大肠杆菌中表达并分泌ScFv抗体。用ELISA检测其对膀胱癌细胞的结合活性。用固定化金属螯合亲和层析（IMAC）在IDA-Ni2+树脂上纯化带有5×组氨酸标签的ScFv抗体。对纯化的ScFv抗体进行SDS-PAGE分析。
VH和VL基因全长分别为366和324个碱基对。与其他已发表序列相比，VH基因是小鼠重链VH亚群II的成员，起源于VH、Dsp2.2和JH4的重排；VL基因是VK亚群IV，来自Vk和Jk4。ScFv抗体可抑制原始单克隆抗体BDI-1 84%的抗原结合活性。纯化的ScFv抗体在SDS-PAGE上呈现单一主要条带（Mr-29 000）。
成功构建并表达了抗人膀胱癌单链Fv抗体。
