【摘要】 根据成熟昆虫神经毒素BjαIT的氨基酸序列,人工合成毒素基因,并克隆至大肠杆菌表达载体pPET-30a(+)。在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物经镍亲和层析纯化,纯化蛋白免疫BALB/c小鼠,制备了特异性较高的抗血清,抗体滴度达1:512,000。提取免疫小鼠脾细胞总RNA,通过RT-PCR分别扩增重链可变区(VH)及轻链可变区(VL)基因片段,在连接肽linker的连接下成功构建了包含抗BjαIT的全长scFv基因库。本研究为新型蝎昆虫神经毒素BjαIT的检测奠定了基础。更多还原

【Abstract】 According to mature toxin amino acid sequence of insect scorpion neurotoxin BjαIT,bjαit gene was synthesized and cloned to vector of PET-30a(+).The fusion protein of BjαIT was expressed in E.coli induced with IPTG and purified with Ni-NTA His Bind Column.After immunization BALB/c mice with purified fusion protein,the high quality antiserum was obtained.The highest titer of the antiserum was about 1:512000.Total RNA was extracted from immunized mouse’s spleen cells.VH and VL were synthesized by RT-PCR with the cDNA of the total RNA.The full long scFv fragments were obtained by linking VH and VL with linker polypeptide.更多还原