抗黄曲霉毒素B1单链抗体基因克隆及其结构分析  

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

作　　者：马兰 刘爱平[2] 王佳[1] 王小红[1] MA Lan;WANG Jia;WANG Xiao-hong;LIU Ai-ping(College of Food Science and Technology,Huazhong Agricultural University,Wuhan 430070,China;College of Food Science,Sichuan Agricultural University,Ya’an,Sichuan 625014,China)

机构地区：[1]华中农业大学食品科技学院,武汉430070 [2]四川农业大学食品学院,四川雅安625014

出　　处：《南方农业学报》2017年第11期2064-2070,共7页Journal of Southern Agriculture

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)

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。【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-AFB1hybridoma cell strains were used as materials,the variable region of heavy chain(VH)and light chain(VL)were amplified by PCR,then anti-AFB1gene scFv was built with(Gly4Ser)3as 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-AFB1gene scFv was744bp in length,encoding248amino acids.The molecular weight was26312.05Da,and theoretical isoelectric point(pI)5.70.In secondary structure,there were35α-helixes(14.11%),28β-folds(11.29%),85extended strands(34.28%)and100random 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.【Conclusion】The constructed anti-AFB1scFv VH contain117amino acids and VL contained116amino acids with a typical antibody structure-groove structure,which can combine with antigen specificity.

关 键 词：黄曲霉毒素B1(AFB1) 单链抗体(scFv) 重链可变区(VH) 轻链可变区(VL) 沟槽结构 

分 类 号：S859.87[农业科学—临床兽医学]