鸭TLR3基因胞外区的原核表达及抗体制备

PROKARYOTIC EXPRESSION AND ANTIBODY PREPARATION OF THE EXTRACELLULAR DOMAIN OF DUCK TOLL-LIKE RECEPTOR 3

采用PCR方法扩增鸭TLR3基因胞外区,将其克隆至原核表达载体p ET-32a(+)中,转化大肠杆菌BL21(DE3)菌株,经IPTG诱导后,表达重组TLR3蛋白,并从包涵体中纯化重组蛋白。结果显示,PCR扩增得到972 bp的TLR3基因胞外区,用纯化的重组TLR3蛋白免疫实验兔,制备TLR3的多克隆抗体,用Western blot和ELISA检测其特异性及效价。本研究成功表达了TLR3基因胞外区,用该蛋白免疫实验兔后,制备的多抗能与TLR3特异性反应。

To study the function of duck TLR3, prokaryotic expression plasmid of Beijing duck Toll-like receptor 3（Toll-like receptor 3, TLR3） gene was constructed and expressed in E. coli BL21（DE3）. The obtained TLR3 protein was purified from inclusion body and its polyclonal antibodies were prepared. The extracellular domain of duck TLR3 gene was amplified with PCR and the resulting 972 bp fragment was cloned into expression vector p ET-32a（＋）. The recombinant plasmid p ET-32a/TLR3 was then transformed into E.coli BL21（DE3） for expression with induction of IPTG. Duck TLR3 protein was purified from the obtained inclusion body. The recombinant protein was used to vaccinate rabbits to prepare polyclonal antibodies. The antiserum samples were collected and examined with Western blot and ELISA, demonstrating its specificity and reactivity.