抗黄曲霉毒素B1单链抗体基因克隆及其结构分析

发布时间：2018-05-23 23:13

本文选题：黄曲霉毒素B(AFB) + 单链抗体(scFv)　；参考：《南方农业学报》2017年11期

【摘要】：【目的】克隆构建黄曲霉毒素B1(AFB1)单链抗体(scFv)基因,并对其编码蛋白序列和结构进行分析预测,为scFv分子修饰改造及在免疫学检测中的应用提供参考依据。【方法】以抗AFB1单克隆抗体杂交瘤细胞株为原料,通过PCR扩增重链可变区(VH)和轻链可变区(VL),以(Gly4Ser)3为柔性接头(Linker)拼接构建抗AFB1 scFv基因,并采用在线生物信息学分析软件对其氨基酸序列、理化性质及蛋白结构进行分析预测。【结果】抗AFB1 scFv基因全长744 bp,编码248个氨基酸,分子量为26312.05 Da,理论等电点(pI)5.70,其二级结构中含有35个α-螺旋(占14.11%)、28个β-折叠(占11.29%)、85个延伸链(占34.28%)和100个随机卷曲(占40.32%),在三级结构中连接肽卷曲将VH和VL区域牵拉而相互靠近,形成典型的抗体结构——沟槽结构,是scFv的抗原结合区域。【结论】构建的抗AFB1 scFv其VH含有117个氨基酸、VL含有116个氨基酸,具备典型抗体结构——沟槽结构,能与抗原特异性结合。
[Abstract]:[objective] to clone and construct aflatoxin B1 AFB1 single chain antibody (scFv1) gene, and to analyze and predict its protein sequence and structure. To provide reference for the modification of scFv molecule and its application in immunological detection. [methods] hybridoma cell lines with monoclonal antibody against AFB1 were used as raw materials. The heavy chain variable region (VH) and the light chain variable region (PCR) were amplified by PCR. The AFB1 scFv resistant gene was constructed by using Gly4Serf3 as the flexible connector. The amino acid sequence was analyzed by online bioinformatics software. Physical and chemical properties and protein structure were analyzed and predicted. [results] the total length of AFB1 scFv gene was 744 BP, encoding 248 amino acids. The molecular weight is 26312.05 Da. the theoretical isoelectric point Pi is 5.70. Its secondary structure contains 35 伪 -helices (14.11), 28 尾 -folds (11.29%), 85 extension chains (34.28%) and 100 random curls (40.32%). In the tertiary structure, the peptide curls draw the VH and VL regions close to each other. The antigen-binding region of scFv is formed by forming a typical antibody structure. [conclusion] the constructed anti-VH has 117 amino acids, and VL contains 116 amino acids, and has a typical antibody structure -groove structure. Can bind to antigen specifically.
【作者单位】：华中农业大学食品科技学院;四川农业大学食品学院;
【基金】：国家自然科学基金项目(31601539) 湖北省自然科学基金项目(2016CFB143) 华中农业大学自主科技创新基金项目(2662015QD027)
【分类号】：R446.6

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