鼠源噬菌体抗体展示库的构建及初步应用

Construction and preliminary application study of phage display antibody library from mouse

构建可用于特异性单链抗体(ScFv)筛选和应用的大容量鼠源噬菌体抗体展示库,获得拥有自主知识产权的抗体库材料。从小鼠脾脏细胞中提取总RNA,反转录成c DNA后,分别扩增抗体的重链基因、轻链基因,并设计2条互补的Linker基因。采用重叠延伸PCR(SOE-PCR)法将重链-Linker和Linker-轻链拼接为完整的ScFv基因,酶切、酶连后,将ScFv基因克隆到pCANTAB-5E噬菌粒载体上,经电穿孔导入E.coli TG1感受态细胞中,过夜培养获得初级噬菌体展示库。以单克隆菌落数计算库容量大小,通过比对随机挑取的单克隆噬菌体ScFv可变区序列的差异,分析库的多样性。以Bt毒素(Cry1B、Cry1C、Cry1F)为包被抗原,从库中筛选具有结合活性的ScFv验证库的实用性。经检测,提取的总RNA条带清晰,浓度为2.76μg/μl,扩增的抗体重链基因、轻链基因条带清晰,且2条互补的Linker基因成功添加到重链基因和轻链基因上。经SOE-PCR法扩增,重链-Linker与Linker-轻链成功拼接为ScFv基因。电转化后,经PCR(Polymerase chain reaction)扩增和凝胶电泳检测,证实ScFv基因克隆成功,测得初级噬菌体抗体展示库的库容为3.67×10~8。随机挑取12个单克隆菌种进行测序和比对,证实ScFv基因为鼠源抗体基因,且12个单克隆菌种的ScFv基因可变区均有一定差异,说明ScFv基因具有多样性。以3种Bt毒素(Cry1B、Cry1C、Cry1F)为抗原,经4轮特异性富集筛选后均获得了具有抗原结合活性的噬菌体ScFv菌种,表明该库的实用性较强。

A large phage display antibody library of mice for ScFvs screening and application research was constructed to obtain the independent intellectual property of the library. Extraction total RNAs from spleen cells of mice,then reverse transcribed them to c DNAs. Amplification of heavy chain gene（ VH） and light chain gene（ VL）fragments with PCR,design two complementary Linker se-quences and add to VH and VL respectively. Splicing VH-Linker and Linker-VL sequences by overlapping extension PCR（ SOE-PCR） to a whole ScFv. Then clone ScFv into the pC ANTAB-5E phagemid vectors,after being digested by restriction endonuclease（ Not I、Sfi I） and linked by T4 DNA ligase. Obtained primary phage display antibody library with over-night cultured that the pC ANTAB-5E-ScFv recombinant plasmid introduced into E. coli TG1 by electroporation. The capacity of this library was calculated by monoclonal colonies. To analyze the diversity of the library,the amino acid sequences of monoclonal colony phage ScFv in variable regions were alignment. We made Bt toxin proteins as coating antigen to verify its practicality by screening ScFvs which had antigen binding activity from the library. After detection,the total RNAs had clear bands and its concentration was 2.76 μg / μl. We could clearly find bands of VH and VL by agarose gel electrophoresis and their complementary Linker genes were added respectively. Finally,the VH-Linker and VL-Linker were spliced to ScFvs by SOE-PCR and cloned into the pC ANTAB-5E phagemid vector successfully. We obtained the primary phage display antibody library with a capacity of 3.67× 108. By sequencing and sequence alignment of 12 monoclonal colonies phage ScFvs from random selection,we could determin the ScFv were from mice and the library had high diversity. After four rounds of panning in the library with three kinds of Bt（ Cry1B、Cry1C、Cry1F）,the ScFvs which could combine with Cry1B、Cry1C、Cry1F were obtained. The results showed that this library had a stronger practicality.