Li Hongbo, Xia Yuxian
Genetic Engineering Research Center, Bioengineering College, Chongqing University, Chongqing 400030, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Oct;24(10):1761-7.
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.
根据巴斯德毕赤酵母的密码子偏好性，基于成熟昆虫神经毒素基因LqhIT2的氨基酸序列进行合成，并分别克隆到PET-30a(+)和pPIC9K载体中。在大肠杆菌中表达的融合蛋白用IPTG诱导并用Ni-NTA His Bind柱纯化。纯化后的融合蛋白用于免疫BALB/c小鼠，获得的抗血清具有高度特异性，效价超过1:128 000。利用该抗血清，通过斑点印迹法筛选出巴斯德毕赤酵母的高表达转化子。在摇瓶中，重组LqhIT2的最高表达量约为9 mg/L。在大肠杆菌中表达的LqhIT2融合蛋白对蝗虫无毒，但在巴斯德毕赤酵母中表达的重组LqhIT2通过注射对蝗虫具有杀虫活性。
