| 巨型引物PCR法对抗CD3改型单链抗体 |
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| 引用本文： | 刘晶,尹长城,黄华梁,姜述德.巨型引物PCR法对抗CD3改型单链抗体[J].生物化学与生物物理进展,2001,28(5):756-760. |
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| 作者姓名： | 刘晶 尹长城 黄华梁 姜述德 |
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| 作者单位： | 1. 中国协和医科大学中国医学科学院医学生物学研究所昆明6501
2. 中国科学院遗传学研究所北京100101 |
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| 基金项目： | 国家“863”资助项目(102-09-04-01). |
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| 摘 要： | 亲和力是影响改型单链抗体应用于临床的重要因素之一.利用巨型引物PCR定点诱变方法,设计并化学合成出两组含多个突变位点的简并引物,在第一轮PCR中使用简并引物分别扩增出含突变碱基的两条特异性的DNA片段,即巨型引物,将其经琼脂糖凝胶电泳分离纯化后,作为3′和5′的两端引物应用于第二轮PCR反应中.通过改变标准PCR反应条件,调整引物与模板的浓度,扩增出特异性较强的目的DNA条带.PCR产物经回收后,进行DNA测序.测序结果表明利用该方法扩增得到特异的抗CD3改型单链抗体的突变体库.
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| 关 键 词： | 改型单链抗体 定点诱变 PCR 巨型引物 慢速退火 |
| 收稿时间： | 2000/11/16 0:00:00 |
| 修稿时间： | 2/9/2001 12:00:00 AM |
Site-directed Mutagenesis of Anti-CD3 Single-chain Fv Fragment(ScFv) Using Two Side Megaprimer PCR Method |
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| LIU Jing,YIN Chang-Cheng,HUANG Hua-Liang and JIANG Shu-De.Site-directed Mutagenesis of Anti-CD3 Single-chain Fv Fragment(ScFv) Using Two Side Megaprimer PCR Method[J].Progress In Biochemistry and Biophysics,2001,28(5):756-760. |
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| Authors: | LIU Jing YIN Chang-Cheng HUANG Hua-Liang and JIANG Shu-De |
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| Institution: | Institute of Medical Biology, Peiking Union Medical College, Chinese Academy of Medical Sciences, Kunming 650118, China;Insitute of Genetics, The Chinese Academy of Sciences, Beijing 100101, China;Insitute of Genetics, The Chinese Academy of Sciences, Beijing 100101, China;Institute of Medical Biology, Peiking Union Medical College, Chinese Academy of Medical Sciences, Kunming 650118, China |
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| Abstract: | In order to improve the affinity of anti CD3 ScFv, one site directed mutagenesis method using "megaprimer" PCR was designed. Adopting the mutagenic primer in the first round of PCR, mutation DNA fragments of 180bp and 160 bp were amplified, respectively. Using these fragments as "megaprimers" in the second round of PCR, specific full length DNA by changing the PCR program and adjusting the concentration of the primer and template were obtained. The sequencing results showed that the mutant pool of anti CD3 ScFv had been yielded successfully. |
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| Keywords: | Anti-CD3 ScFv site-directed mutagenesis polymerase chain reaction megaprimer slow annealing |
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