| 抗红斑丹毒丝菌的rSpaA蛋白猪源单链抗体库构建及单链抗体淘选 |
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| 引用本文： | 王锐,葛猛,陈一苇,方超,柴强强,李润成,余兴龙.抗红斑丹毒丝菌的rSpaA蛋白猪源单链抗体库构建及单链抗体淘选[J].湖南农业大学学报(自然科学版),2016,42(6):647-653. |
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| 作者姓名： | 王锐 葛猛 陈一苇 方超 柴强强 李润成 余兴龙 |
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| 作者单位： | 湖南农业大学动物医学院,湖南长沙,410128 |
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| 基金项目： | 国家自然科学基金项目(31272652)；现代农业产业技术体系建设专项(CARS-46-42) |
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| 摘 要： | 从免疫过rSpaA的猪的脾淋巴细胞中提取总RNA,采用RT–PCR技术反转录合成cDNA。设计兼并引物扩增抗体重链可变区(VH)和轻链可变区(VL)基因片段,采用重叠延伸PCR方法,将VH和VL通过Linker连接成重组单链抗体(以下简写为sc Fv)的基因片段。将sc Fv的基因连接至噬菌粒载体p Comb3Xss,将重组载体电转化至宿主菌XL1-Blue,并经辅助噬菌体M13KO7拯救,获得猪源噬菌体单链抗体库,库容约为2.5×106。以rSpaA为靶抗原,经免疫亲和筛选,获得8株特异性较好的阳性克隆。本研究结果可为制备抗红斑丹毒丝菌的重组sc Fv提供新途径,并为猪丹毒的免疫检测和综合防制提供材料基础。
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| 关 键 词： | 猪源抗体库 单链抗体 红斑丹毒丝菌 噬菌体展示技术 |
Construction of swine phage single-chain antibody variable fragment (scFv) library for recombinant SpaA protein (rSpaA)of erysipelothrix rhuriopathiae and specific scFv panning |
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| Abstract: | The study was designed to identify and construct a library for swine phage single-chain antibody (scFv) rSpaA. Total RNA was extracted from splenic tissue and used to amplify fragments consisting of a variable heavy chain (VH) and a variable light chain (VL). After purification, VH and VL were joined with a linker to produce scFv fragments by splicing-overlap-extension (SOE) PCR. The gene scFv was cloned in plasmid pComb3Xss, and the recombinant phagemids were transformed to susceptibleE. coli XL1–Blue. After infection with the aid of phage M13K07, the library of phage antibody was constructed. The size of constructed antibody library was 2.5×106. By taking the purified recombinant protein rSpaA as target antigens, 8 strains of positive scFvs with binding affinity to rSpaA were identified after 5 rounds of procedure of ELISA: bind-elution-enrichment. In conclusion, the construction of rSpaA swine scFv library and the study for the preparation of recombinant antibodies could provide a new way and lay the foundation for the immunoassay of SE and comprehensive prevention and control to swine erysipelas. |
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| Keywords: | swine antibody library single-chain antibody (scFv) phage library display erysipelothrix rhuriopathiae |
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