BjαIT的表达、抗血清制备及scFv基因库的构建

Expression,antiserum preparation and scFv gene library construction of BjαIT

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。

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.