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。
The mRNAs of spleens from mice with a high titer of antibody against Clostridium perfringens were extracted,two fragments of VH about 340 bp and VL about 325 bp were obtained through RT-PCR,and the VH-Linker-VL（ScFv） fragment of 750 bp was obtained through SOE： PCR. The ScFv genes were ligated into the phagemid vector pCANTABSE by using One Step Cloning Kit,and the phage-display library consisted of 3.5 × 10^6 clones. Affinity enrichment screening of phage-ScFv surface display library with Ia,Ib as antigen were conducted five times,the positive rate reached about 70%. Three strongest screened positive affinity antibodies （Ia-4, Ia-8 and Ibl） were expressed as soluble, this experiment constructed successfully the single-chain phage antibody against Ia and Ib of ι toxin,which made a foundation of specific antibody mass production of toxin,also laid a theoretical value and practical significance for detection and clinical treatment of Clostridium perfringens ι toxin infections.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2017年第2期243-249,共7页
Chinese Journal of Veterinary Science
基金
河北省科技支撑计划资助项目(13226603D)
