抗人宫颈癌单链抗体的表达、结构预测与功能分析.doc

抗人宫颈癌单链抗体的表达、结构预测和功能分析 作者：王莹；李旭；陈葳　　(西安交通大学第一医院医学实验中心，陕西??西安??710061) 摘要：目的??扩增、表达抗人宫颈癌单链抗体 (ScFv)基因；对ScFv蛋白进行活性鉴定、二级结构、三维结构预测和理化性质分析。方法??采用基因重组技术构建抗人宫颈癌ScFv基因，进行TG1和HB2151两个原核体系的表达；SDS、Western blotting、竞争抑制实验、免疫组化反应检测ScFv；用Antheprot 4.3软件、Swiss-model、3dpssm 进行蛋白理化性质分析和立体结构模拟。结果??抗人宫颈癌可溶性ScFv大小为32 000左右，与预计蛋白相对分子质量相符；可溶性和展示性ScFv均可与人宫颈癌细胞株细胞膜表面抗原结合。免疫组化显示能与宫颈癌组织特异性结合而不与正常宫颈组织和其他肿瘤组织结合。ScFv蛋白等电点预测值为7.215，属于α+β蛋白；VH和VL区均有多个蛋白激酶C磷酸化位点和酪氨酸激酶II磷酸化位点。三维结构预测显示linker 贴近，使VL和VH形成一个疏水的“口袋”，这一结构有利于抗原的结合。结论　经基因工程方法制备的抗人宫颈癌ScFv具有与亲本单克隆抗体相似的抗体活性和特异性。抗人宫颈癌ScFv构建为进一步构建双特异性ScFv和双功能ScFv基因创造了条件；其成功的表达为人宫颈癌的早期诊断和特异性治疗打下了基础。计算机对ScFv蛋白的空间结构的模拟和理化性质的分析为ScFv的进一步改建和抗原、抗体反应的研究提供了宝贵的资料。 关键词：宫颈癌；单链抗体；二级结构；三维结构 中图分类号：R737.33??文献标识码：A??文章编号：1673-4254(2006)01-0016-06 Expression, structure prediction and functional analysis of murine single-chain fragment variable antibody against human cervical cancerWANG Ying；LI Xu；CHEN WeiMedical Laboratory Center, First Hospital of Xi’an Jiaotong University, Xi’an 710061, ChinaAbstract: Objective To amplify and express the gene encoding murine single-chain fragment variable (scFv) antibody against human cervical cancer and predict the secondary structure and three-dimensional structure of the antibody. Methods The gene fragments coding for the variable region of the heavy and light chains of scFv antibody against human cervical cancer were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, and spliced together using a flexible linker to the antibody. The scFv genes were then cloned into the expression vector pCANTAB 5E and expressed in E.coli HB2151 and TG1, respectively. The scFv antibody obtained was assayed using SDS, Western blotting, and immunohistochemical analysis, with its secondary structure and three-dimensional structure predicted with Swiss-model and 3dpssm. The physicochemical properties of the antibody were analyzed with Antheprot software. Results The expressed scFv antibody was soluble and phage-displaye