昆虫神经毒素LqhIT2的表达、抗血清制备及活性分析

Expression, Antiserum Preparation and Bioactivity Assays of Insect Neurotoxin LqhIT2

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, 摇甁条件下,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。

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 E. 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 Escherichia coli was not toxic to locust, but the recombinant LqhIT2 expressed in P. pastoris had insecticidal activity against locust through injection.