【摘要】 根据毕赤酵母密码子偏爱性,不改变毒素蛋白质一级结构,设计合成了昆虫神经毒素LqhIT2基因,并分别克隆至大肠杆菌融合表达载体pPET30-a(+)和毕赤酵母分泌表达载体pPIC9K。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后,用于免疫BALB/c小鼠,制备了特异性较高的抗血清,抗体滴度超过1:128 000。利用制备的抗血清,采用斑点杂交,筛选得到了较高水平分泌表达重组LqhIT2的酵母转化子,摇摇摇摇下,毒素表达量约9 mg/L。大肠杆菌表达产物没有生物活性,酵母表达产物经注射蝗虫表现出杀虫活性。更多还原

【Abstract】 According to the codon bias of Pichia pastoris,the mature insect neurotoxin gene LqhIT2 was synthesized based on its amino acid sequence and was cloned to vector of PET-30a(+) and pPIC9K respectively.The fusion protein expressed in Escherichia.coli was induced with IPTG and purified with Ni-NTA His Bind Column.The purified fusion protein was used to immunize BALB/c mice,and antiserum obtained was highly specific with the titer of over 1:128 000.Using the antiserum,high-level expression transformants of P.pastoris were screened by dot blotting.The highest expression of recombinant LqhIT2 was about 9 mg/L in baffled flasks.The fusion protein of LqhIT2 expressed in E.coli was not toxic to locust,but the recombinant LqhIT2 expressed in P.pastoris had insecticidal activity against locust through injection.更多还原