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

发布时间：2018-11-09 21:23

【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。
[Abstract]:Extraction of total RNA, from spleen lymphocytes of pigs immunized with rSpaA and reverse transcription synthesis of cDNA. using RT PCR technique Degenerate primers were designed to amplify the fragments of heavy chain variable region (VH) and light chain variable region (VL) of antibody. By means of overlapping extension PCR, VH and VL were linked with Linker to form recombinant single chain antibody (hereinafter referred to as sc Fv) gene fragment). The sc Fv gene was ligated to the phagocyte vector p Comb3Xss,. The recombinant vector was electrotransformed into the host strain XL1-Blue, and rescued by auxiliary bacteriophage M13KO7. The porcine phage scFv library was obtained and the library capacity was about 2.5 脳 106. Using rSpaA as the target antigen, 8 positive clones with good specificity were obtained by immuno-affinity screening. The results can provide a new way for the preparation of recombinant sc Fv against erysipelas and provide a material basis for the immunoassay and comprehensive control of porcine erysipelas.
【作者单位】：湖南农业大学动物医学院;
【基金】：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)
【分类号】：S858.28

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