抗重组猪蛔虫抗原AD41蛋白鼠源性单链抗体库的构建及筛选

为了构建重组猪蛔虫抗原AD41蛋白鼠源性单链抗体库,试验用重组蛋白AD41抗原免疫接种Balb/c小鼠,提取小鼠脾脏总RNA,以总RNA为模板经RT-PCR合成cDNA,再以cDNA为模板,用小鼠免疫球蛋白重链和轻链设计引物,分别扩增重链和轻链可变区基因,利用SOE-PCR法将VH和VL片段随机拼接成单链抗体(ScFv)片段,将其克隆入质粒表达载体pCANTAB5E中,转化于大肠杆菌TG1,通过辅助噬菌体M13K07援救构建噬菌体单链抗体库。结果表明:从30个噬菌体克隆中筛选到25个阳性克隆。说明成功地构建了抗重组猪蛔虫抗原AD41蛋白鼠源性单链抗体库。

Construction and screening of single-chain antibody library associated with recombinant AD41 antigen from Ascaris suum

Balb/c mice were injected with the recombinant protein AD41 of Ascaris suum mixed with FCA.Total RNA was extracted from the spleen cell of the immune Balb/c mouse through RT-PCR.The immunoglobulin heavy-chain and light-chain variable region genes(VH and VL) repertoire were amplified respectively by PCR.Then they were linked by SOE(splicing by overlap extension) PCR.These fragments were cloned into the phagemid pCANTAB5E and the recombinant phagemids were transformed into susceptible Escherichia coli TG1 by electroporation.The single-chain library was constructed after rescuing with helper phage M13K07.The results showed that twenty-five antigen binding clones were identified from 30 individual phagemid clones.The single-chain antibody library associated with recombinant AD41 antigen from Ascaris suum was successfully constructed.