Synergy Between Aedes aegypti Trypsin Modulating Oostatic Factor and δ-Endotoxins

D. Borovsky, V. Khasdan, S. Nauwelaers, Clara Theunis, Lien Bertie r, E. Ben‐Dov, A. Zaritsky
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引用次数: 1

Abstract

Starved first instar Aedes aegypti larvae were 35-fold more sensitive to Bacillus thuringiensis subsp. israelensis (Bti) toxins than fed larvae. Feeding larvae Pichia pastoris yeast cells expressing tmfA (synthetic gene coding for the Trypsin Modulating Oostatic Factor of Ae. aegypti) together with Escherichia coli cells expressing Bti toxin genes (cry4Aa, cry11Aa, cyt1Aa and p20) indicate that TMOF and Cry toxins are synergisitic. tmfA was cloned and expressed in the cyanobacterium Anabaena PCC 7120 and the hormone was purified by HPLC and identified by ELISA. The amount of TMOF synthesized by Anabaena was low (0.5 - 1 μg in 10 8 cells). P. pastoris, which synthesizes high amounts of heterologous proteins in the presence of methanol and is readily consumed by mosquito larvae, was genetically engineered to produce more TMOF. Codon-optimized synthetic genes, cry11Aa- tmfA and gst-cry11Aa- tmfA, that were cloned into P. pastoris and fed to Ae. aegypti larvae caused 87.5% mortality in 5 days. GST (glutathione-S-transferase) enhanced the activity of Cry11A-TMOF and protected it from heat denaturation. Cell free extracts of recombinant P. pastoris cells killed 40% of tested 4 th instar larvae within 24 h, and mass spectra analysis confirmed that the recombinants synthesize Cry11Aa. This report shows for the first time that Cry toxins and TMOF are synergists to Ae. aegypti larvae when jointly fed or expressed in recombinant P. pastoris.
埃及伊蚊胰蛋白酶调节分泌因子与δ-内毒素的协同作用
饥饿的1龄埃及伊蚊幼虫对苏云金芽孢杆菌的敏感性提高35倍。以色列蚊(Bti)毒素比饵料幼虫多。表达胰酶调节泌乳因子合成基因tmfA的毕赤酵母细胞喂养幼虫。埃及伊蚊)和表达Bti毒素基因(cry4Aa、cry11Aa、cyt1Aa和p20)的大肠杆菌细胞表明TMOF和Cry毒素具有协同作用。克隆了tmfA并在蓝藻Anabaena PCC 7120中表达,通过HPLC纯化,ELISA鉴定。赤霉素合成量低(10 8个细胞0.5 ~ 1 μg)。巴斯德梭菌在甲醇的存在下可以合成大量的异源蛋白,并且很容易被蚊子幼虫消耗,通过基因工程使其产生更多的TMOF。经密码子优化的合成基因cry11Aa- tmfA和gst-cry11Aa- tmfA克隆到P. pastoris中,并喂给Ae。埃及伊蚊幼虫5 d死亡率为87.5%。GST(谷胱甘肽- s -转移酶)增强了Cry11A-TMOF的活性,并对其热变性具有保护作用。重组pastoris细胞的无细胞提取物在24 h内杀死了40%的4龄幼虫,质谱分析证实重组蛋白能合成Cry11Aa。本报告首次表明,Cry毒素和tof对Ae具有增效作用。埃及伊蚊幼虫联合喂养或重组帕斯德酵母表达。
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