抗人VEGF单链抗体基因的克隆及突变校正  

Cloning and reverse mutation of a single chain Fv antibody against human VEGF

作　　者：廖楚航[1] 王大章[2] 杨西川[2] 李扬[2] 房思炼[2] 

机构地区：[1]四川省人民医院口腔颌面外科,成都610072 [2]四川大学华西口腔医学院口腔颌面外科,成都610041

出　　处：《实用医院临床杂志》2004年第1期26-29,共4页Practical Journal of Clinical Medicine

基　　金：国家自然科学基金(39970798)

摘　　要：目的:回复基因克隆过程中的异常点突变,重新表达抗人血管内皮生长因子(VEGF)单链抗体基因(V11),恢复其抗原结合能力。方法:通过逆转录及多聚酶链式反应(RT-PCR),扩增抗人VEGF单克隆抗体(E11)的可变区基因片段,构建表达载体pET-15b(含E-tag)。将V11轻、重链可变区基因序列(V11L、V11H)与其所属亚组共同序列和一致序列进行联配比较,发现异常氨基酸并分析其性质,确定异常突变点。PCR定点回复突变,重新表达单链抗体基因(V11m),ELISA检测表达产物与VEGF165的结合。结果:V11存在四个异常突变点:V11L第7、23、36位,V11H第113位。回复突变后的表达产物与VEGF165的结合能力显著提高。结论:利用抗体残基位置序列保守性及氨基酸侧链性质分析,可以推定并有助于回复异常点突变,该方法简便易行。获得的抗人VEGF单链抗体对恶性肿瘤的诊治具有潜在的应用价值。Objective: To resume the binding ability of a single chain Fv (ScFv) antibody against human VEGF by revising the point mutation and expressing it over again. Methods: From the hybridoma cell line secreting monoclonal antibody against VEGF, total RNA was prepared and used as a template for cDNA synthesis and cloning. Reverse transcription and polymerase chain reaction (RT-PCR) were used to clone the immunoglobulin variable region (V11H ,V11L ). The constructed ScFv was inserted into pET-15b with a E-tag at the 3'end. Compared with its commom and consensus sequence, the point mutations were confirmed and then revised by PCR. The binding activity of the revised ScFv was evaluated by ELJSA. Results: 4 point mutations were found in sequence of ScFv and the revised ScFv had a higher binding ability to VEGF 165 than mutated one. Conclusion: Based upon analyse of the conservative sequence of the amino residues and its property , it is a simple and convenient way to confirm the point mutations. The revised SvFv is potentially useful in both diagnostic and therapeutic application of human malignant tumors.

关 键 词：血管内皮生长因子 恶性肿瘤 单链抗体基因 基因克隆 突变校正 

分 类 号：R730.54[医药卫生—肿瘤]