抗黄曲霉毒素B1单链抗体基因克隆及其结构分析

【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。

Anti-aflatoxin B1 single chain antibody:Gene cloning and structure analyzing

Objective]In this work,the anti-aflatoxin B1(AFB1)single chain antibody(scFv)gene was constructed, and its encoded protein sequence and structure was analyzed and predicted,which provided reference for scFv molecular modification and application in immunological detection.Method]In this study,anti-AFB1 hybridoma cell strains were used as materials,the variable region of heavy chain(VH)and light chain(VL)were amplified by PCR,then anti-AFB1 gene scFv was built with(Gly4Ser)3 as flexible joint(Linker). The amino acid sequence,its physicochemical properties and protein structure were analyzed by online bioinformatics analysis software.Result]The results indicated that the full anti-AFB1 gene scFv was 744 bp in length,encoding 248 amino acids. The molecular weight was 26312.05 Da,and theoretical isoelectric point(pI)5.70. In secondary structure,there were 35α-helixes(14.11%),28β-folds(11.29%),85 extended strands(34.28%)and 100 random coils(40.32%). In tertiary structure,connective peptides pulled together VH and VL to form a typical abtibidu structure—groove structure,which was the antigen binding region for scFv.Conclu-sion]The constructed anti-AFB1 scFv VH contain 117 amino acids and VL contained 116 amino acids with a typical antibody structure—groove structure,which can combine with antigen specificity.