抗产气荚膜梭菌ι毒素Phage-ScFv库的构建与筛选

发布时间：2018-03-12 15:00

  本文选题：产气荚膜梭菌ι毒素　切入点：Ia　出处：《中国兽医学报》2017年02期 　论文类型：期刊论文

【摘要】：选取经产气荚膜梭菌ι毒素免疫且抗体效价较高的小鼠,提取脾脏mRNA,通过PT-PC技术,分别获得340,325bp的重链可变区基因(VH)和轻链可变区基因(VL)。利用重叠延伸PCR(SOE-PCR)获得大小为750bp的VH-Linker-VL单链抗体基因(ScFv)。利用One Step Cloning Kit试剂盒将ScFv基因片段与噬菌粒载体pCANTAB5E连接,构建库容约为3.5×106抗ι毒素噬菌体初级抗体库。以Ia、Ib为抗原进行Phage-ScFv表面展示文库的5轮亲和富集筛选,最终阳性率均可达70%。选取亲和力最强的3株阳性抗体(Ia-4、Ia-8和Ib1)进行可溶性表达,为ι毒素特异性抗体的大量制备奠定基础,并对产气荚膜梭菌ι毒素感染病例的检测和临床治疗研究具有一定的理论价值和实践意义。
[Abstract]:Selection of Clostridium perfringens iota toxin in immune and high titers of mice spleen extract, mRNA, through PT-PC technology, heavy chain variable region gene were obtained with 340325bp (VH) gene and the variable region of light chain (VL). Using overlap extension PCR (SOE-PCR) to obtain the size of VH-Linker-VL scFv gene 750bp (ScFv) the use of One Step. Cloning Kit kit for connecting the ScFv gene fragment and phagemid vector pCANTAB5E, construct the capacity of about 3.5 x 106 l anti phage antibody library. The primary toxin Ia, Ib antigen in Phage-ScFv surface display library of 5 rounds of affinity enrichment screening, the final positive rate was up to 70%. from the highest affinity 3 strains of positive antibody (Ia-4, Ia-8 and Ib1) soluble expression system for a large number of iota toxin specific antibody preparation and lay the foundation for C.perfringens iota toxin infection detection and clinical research has certain The theoretical value and practical significance.

【作者单位】： 河北师范大学生命科学学院;
【基金】：河北省科技支撑计划资助项目(13226603D)
【分类号】：S852.61
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本文编号：1602068