抗ICOSL核糖体展示单链抗体库的构建  

Construction of Anti-ICOSL Ribosome Display Library

作　　者：严浩[1] 王芳[1] 毛伟平[1] 王文倩[1] 孟素芳 邵峦峦 刘小綪 

机构地区：[1]南京师范大学生命科学学院江苏省分子医学重点实验室,江苏南京210046

出　　处：《安徽农业科学》2013年第33期12819-12822,共4页Journal of Anhui Agricultural Sciences

摘　　要：[目的]构建出鼠源抗ICOSL核糖体展示抗体库。[方法]通过RT-PCR从人B淋巴瘤细胞Daudi的总RNA中扩增出ICOSL胞外区基因,并将此基因插入到原核表达载体pET28a中进行表达。以表达纯化的ICOSL蛋白为抗原免疫Balb/c小鼠,取小鼠脾脏提取总RNA,通过RT-PCR扩增小鼠的重链可变区和Linker(VH-linker)序列,以及轻链可变区和Linker(Vκ-linker)序列。经重组PCR将两段基因连接起来,将VH/κ与pMD19-T载体链接产物转化E.coli DH 5α,PCR鉴定并测序。[结果]VH和κ链得到正确的扩增,长度分别为421和689 bp,VH/κ连接产物正确,连接产物长度为1 079 bp。[结论]所采用的引物及反应条件能够扩增出目的基因,成功构建了鼠源抗ICOSL核糖体展示抗体库,为进行核糖体展示体外筛选抗ICOSL特异性单链抗体奠定基础。[ Objective ] Anti-ICOSL ribosome display library was constructed. [ Method ] The eDNA of ICOSL extracellular domain was ampli- fied from Daudi cell by RT-PCR. The ICOSL gene was inserted into plasmid pET28a to express. RNA from the spleens of Baib/e mice which had been immunized with ICOSL protein were selected . Heavy chain and κ chain genes （ VH and κ） were amplified separately by reverse transcriptase polymerase chain reaction （RT-PCR） , and the anti-ICOSL VH/κ chain ribosome display library was constructed by assembling VH and κ with a special flexible linker by overlap extension PCR （SOE-PCR）. The VH/κ chain was ligated into pMD19-T vector and the ligated sample was transformed into competent E. coli DH5a. The plasmid DNA was identified with PCR and sequenced. [ Results] VH and κ were amplified correctly and the length were 421 and 689 bp respectively. VH/κ was ligated correctly and the length was 1093 bp. [ Conclusion ] The primers and reaction condition were able to generate required genes, VH/κ chain ribosome display library was constructed correctly. It was the basis for screening single chain antibody specific for ICOSL by ribosome display technique.

关 键 词：ICOSL(可诱导共刺激因子配体) 单链抗体 核糖体展示技术 原核表达 

分 类 号：S188[农业科学—农业基础科学]