抗猪流感噬菌体单链抗体库的构建与筛选  

Construction and screening of anti-swine influenza virus phage display scFv library

作　　者：杨康[1,2] 蒋蔚[1] 刘迎春[1] 陈永军[1] 孙卫东[2] 宋宁宁[1] 王权[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2012年第6期21-25,共5页Animal Husbandry & Veterinary Medicine

基　　金：转基因生物新品种培育重大专项(2009ZX08010-022B)

摘　　要：利用噬菌体展示技术构建猪流感病毒噬菌体抗体库,并筛选出猪流感高特异性、高亲和力的单链抗体(scFv)。以猪流感病毒免疫BALB/c小鼠,提取脾细胞总RNA,反转录后以cDNA为模板扩增获得VH基因和VL基因,并采用重叠延伸PCR(SOE-PCR),用柔性多肽Linker接头(Gly4Ser)按VH-Linker-VL方式将VH基因和VL基因拼接成scFv基因片段。将scFv基因和pCANTAB5E载体分别双酶切(SfiⅠ/NotⅠ)后连接,转化宿主菌TG1,经过辅助噬菌体M13K07拯救,构建噬菌体单链抗体库。以猪流感病毒为抗原包被96孔酶标板,经过3轮的亲和富集筛选,用Phage-ELISA鉴定阳性重组抗体。本研究成功构建出库容约为4×106cfu/mL抗猪流感病毒的单链抗体库,并筛选出4株特异性抗猪流感病毒的scFv抗体,能够与鼠源阳性多抗进行竞争结合猪流感病毒,为抗猪流感病毒转基因猪的研究奠定基础。The single chain variable fragment （scFv） antibody library specific to swine influenza virus （SIV） was constructed in this stud- y. The BALB/c mice were immunized with SIV, and the total RNA was extracted from spleen cells and reverse transcribed into cDNA. Using cDNA as template, the variable region genes （VH and VL） from heavy chain and light chain were amplified by PCR, respectively. Then they were linked by a DNA linker （Gly4 Ser） to VH-linker-VL sequence, thus forming seFv by SOE （splicing by overlap extension） PCR. The scFv gene was digested by restriction enzyme Sfi I and Not I , then ligated into the vector of pCANTAB5E. The recombinant vector was transformed into Escherichia coli TG1, and then rescued with the help of phage M13K07. Therefore a library of phage scFv antibody was con- structed. After three rounds of enrichment screening and identification of Phage-ELISA, four strains of scFv antibodies to SIV were obtained from the library. This study provides a basis for the production of transgenic pigs against SIV.

关 键 词：猪流感病毒 噬菌体展示技术 单链抗体 

分 类 号：S852.65[农业科学—基础兽医学]