抗弓形虫噬菌体单链抗体库的构建及筛选

Construction and screening of single-chain antibody library of Toxoplasma gondii

采用弓形虫可溶性抗原攻击的小鼠,取小鼠脾脏从中提取细胞总RNA,通过RT-PCR扩增鼠源抗体VH和VL基因,并采用重叠PCR(SOE-PCR)方法构建ScFv基因,将其克隆入噬粒载体pCANTAB5E中,转化于感受态大肠杆菌TG1,通过辅助噬菌体M13K07援救构建噬菌体单链抗体库.从20个噬菌体克隆中筛选到15个具有弓形虫可溶性抗原结合活性的阳性克隆.研究提示成功地构建出了一个有效库容量为2.2×106的鼠抗弓形虫噬菌体单链抗体库.

A few of mice were injected Toxoplasma gondii RH, their spleen we minced with a surgical knife, total RNA was extracted. To mice immunoglobulin H chain and L chain variable region of degenerate primers, eDNA first strand as a template, were amplified H chain and L chain variable region genes. SOE-PCR method to VH and VL fragments were assembled into ScFv fragments and then cloned into pCANTABSE ScFv vector and transformed into competent TG1 by roporation. After rescuing with helper phage M13 K07. Fifteen antigen binding clones were identified from 20 individual phagemid clones. The study showed a 2. 2 × 10^6 murine anti Toxoplasma gondii single chain phage antibody library was successfully constructed.