[1]刘金凤,吴健敏,覃绍敏,等.抗猪瘟病毒单链抗体基因的构建及其推导的蛋白质三级结构的分子模拟[J].江苏农业学报,2015,(04):817-823.[doi:10.3969/j.issn.1000-4440.2015.04.017]
　LIU Jin-feng,WU Jian-min,QIN Shao-min,et al.Construction of single chain antibody gene of anti-CSFV and homology modeling of 3D structure of deduced protein[J].,2015,(04):817-823.[doi:10.3969/j.issn.1000-4440.2015.04.017]
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抗猪瘟病毒单链抗体基因的构建及其推导的蛋白质三级结构的分子模拟()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年04期
页码:
817-823
栏目:
畜牧兽医·水产养殖
出版日期:
2015-08-31

文章信息/Info

Title:
Construction of single chain antibody gene of anti-CSFV and homology modeling of 3D structure of deduced protein
作者:
刘金凤; 吴健敏; 覃绍敏; 白安斌; 陈凤莲; 曹颖颖
（广西壮族自治区兽医研究所，广西南宁530001）
Author(s):
LIU Jin-feng; WU Jian-min; QIN Shao-min; BAI An-bin; CHEN Feng-lian; CAO Ying-ying
(Guangxi Veterinary Research Institute, Nanning 530001, China)
关键词:
猪瘟病毒; 单链抗体; 序列分析; 分子模拟
Keywords:
classical swine fever virus （CSFV）; single-chain variable fragment（ScFv）; sequence analysis; homology modeling
分类号:
S852.65+1
DOI:
10.3969/j.issn.1000-4440.2015.04.017
文献标志码:
A
摘要:
为利用抗猪瘟病毒（Classical swine fever virus，CSFV）单链抗体基因（ScFv）建立CSFV病原快速检测方法，以抗猪瘟病毒单克隆抗体杂交瘤细胞株总RNA为模板，通过RT-PCR扩增重链可变区基因（VH）和轻链可变区基因（VL），以柔性多肽（Gly4Ser）3为接头（Linker），经重叠延伸反应将VH、VL基因拼接为完整的CSFV-ScFv基因，并进行克隆、测序分析。结果显示，所克隆单链抗体基因全长732 bp，包括363 bp的VH、45 bp的Linker和324 bp的VL，为VH-Linker-VL结构，编码的氨基酸序列含有明确的互补决定区（CDR）和框架区（FR）以及特征性的半胱氨酸残基，并与多种鼠源单链抗体基因高度同源，具有重组功能性鼠源抗体可变区特征。对CSFV-ScFv基因所编码蛋白质三级结构进行预测，显示其形成口袋样形状的空间构象，理论上具有良好的抗原结合活性。表明本研究成功构建了CSFV-ScFv基因。
Abstract:
To establish a quick detection assay for classical swine fever virus （CSFV）by using single-chain fragment variable (ScFv) gene, total RNA was first abstracted from the hybridoma cell anti-CSFV14 which could secrete anti-CSFV antibody in order to amplify VH and VL genes in variable regions by RT-PCR. Next, with the flexible polypeptide Gly4Ser3 as the linker, the VL and VH genes were spliced into a full length of CSFV-ScFv by means of splicing overlap extension PCR. Then, the full length of CSFV-ScFv was sequenced following cloning. The cloned CSFV-ScFv gene showed a structure of VH-Linker-VL, with a full length of 732 bp, including the VH of 363 bp, the linker of 45 bp and the VL of 324 bp. It was featured by an explicit complementarity determining region (CDR), a framework region (FR) and characteristic cysteine residues, and was highly homologous to various mouse-borne single-chain genes, indicative of the characteristics of functionally recombinant mouse-borne antibody variable fragments. The computer graphic modeling of its protein 3D structure predicted a pocket-shaped spatial configuration, suggestive of a strong antigen binding activity theoretically. The results demonstrated the successful construction of CSFV-ScFv gene in this research.

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备注/Memo

备注/Memo:
收稿日期：2014-12-10 基金项目：广西科技攻关与新产品试制项目（桂科攻1123007-3） 作者简介：刘金凤 (1986-)，女，广西容县人，硕士，研究实习员，从事动物传染病与分子免疫学研究。（Tel）13407723103;(E-mail)fjinliu@163.com 通讯作者：吴健敏， (E-mail)wu-jm20@163.com
更新日期/Last Update: 2015-08-31