ANEPⅢ毒蛋白的表达调控及其抗虫效果的生物测定

doi:10.16409/j.cnki.2095-039x.2018.03.020

摘要/Abstract

摘要： 蝎体内产生一系列毒素蛋白，ANEPⅢ是其中一种。试验构建重组表达载体pPIC9K-ANEPⅢ，将质粒pNJUTRXA-ANEPⅢ转化大肠杆菌BL21，通过IPTG诱导目标蛋白ANEPⅢ，并用Tricine-SDS-PAGE电泳进行定性分析，检测得到8000 kD目的蛋白。通过电转化的方法将pPIC9K-ANEPⅢ转入毕赤酵母宿主菌GS115中，筛选得到一株耐受2.0 mg/mL G418表型为His⁺Mut⁺的菌株。经0.5%的甲醇诱导，取不同诱导时间的表达上清液进行Tricine-SDS-PAGE电泳分析，确认7800 kD毒性目的蛋白已经获得分泌表达，在诱导72 h时表达量最高。对ANEPⅢ基因的原核与真核表达产物进行提取、初步纯化后进行了抗虫试验。结果表明，原核表达产物没有显示出生物活性，真核表达产物具有较好的生物学活性。浓度为1 mg/mL的真核表达产物，喂食舞毒蛾2龄幼虫5 d后，校正死亡率达37.9%，幼虫食叶量下降50%。黄粉虫的生物测定结果表明，喂食1 mg/mL的真核表达产物5 d后，黄粉虫幼虫的校正死亡率为20%。

关键词: 抗神经兴奋肽, 载体构建, 蛋白表达, 生物测定

Abstract: A series of toxin protein are created in Scorpion's body, anti-neuroexcitation peptide Ⅲ is one of them. The recombinant expression vector pPIC9K-ANEPⅢ was constructed, the plasmid pNJUTRXA-ANEPⅢ was transformed into BL21. The target protein was induced by IPTG, and the expressed protein was evaluated by Tricine-SDS-PAGE. The result indicated that the size of the fusion protein was about 8000 kD. The recombinant plasmid pPIC9K-ANEPⅢ was transformed into Pichia pastoris GS 115 by electro transformation, one of His⁺Mut⁺ strain which can survive on the 2.0 mg/mL G418 plate for expression was screened out. After inducing by 0.5% methanol, the expressed protein has been evaluated by Tricine-SDS-PAGE, the size of the secreted protein was about 7800 kD, and the highest expression level appeared at 72 h. Prokaryotic and eukaryotic expression product of ANEPⅢ gene was extracted, the insect resistance test was carried out after preliminary purification. The result showed that the prokaryotic expression product did not show biological activity, but the eukaryotic expression product had better biological activity. 2nd instar larvae of Lymantria dispar were feeded by eukaryotic expression product, which concentration was 1 mg/mL, the corrected mortality was 37.9% after 5 days, and the larval feeding capacity descended to 50%. The same experiment performed on larvae of Tenebrio molitor by eukaryotic expression product, its concentration was 1 mg·ml^-1, the corrected mortality of larvae was 20.0% after 5 days.

Key words: anti-neuroexcitation peptide, vector construction, protein expression, bioassay

中图分类号:

S476

王占斌, 李勍, 严善春, 冯连荣. ANEPⅢ毒蛋白的表达调控及其抗虫效果的生物测定[J]. 中国生物防治学报, 2018, 34(3): 480-487.

WANG Zhanbin, LI Qing, YAN Shanchun, FENG Lianrong. ANEPⅢ Gene Expression Regulation and Anti-insect Bioassay[J]. journal1, 2018, 34(3): 480-487.

参考文献

[1] Elliott K A, van Gelder N M. Occlusion and metabolism of gamma-aminobutyric acid by brain tissue[J]. Journal of Neurochemistry, 1958, 3(l):28-40.
[2] Iversen L L, Kelly J S. Uptake and metabolism of gamma-aminobutyric acid by neurones and glial cells[J] Biochemical Pharmacology, 1975, 24(9):933-938.
[3] Song Y B, Huang T T, Lai L L, et al. Expression of anti-neuroexcitation peptide Ⅲ of scorpion Buthus martensii Karsch BmK ANEP Ⅲ in plants[J]. Molecular Biology (Mosk), 2011, 45(6):949-955.
[4] 毛力群, 郭三堆. 昆虫神经毒素基因工程研究进展[J]. 生物技术通报, 1998(6):1-6.
[5] 吉永华. 蝎毒中的昆虫神经毒素的生化研究[J]. 生物化学与生物物理进展, 1990, 17(2):100-105.
[6] Schousboe A. Pharmacological and functional characterization of astrocytic gaba transport:A short review[J]. Neurochemal Research, 2000, 25(9/10):1241-1244.
[7] Corona M, Coronas F V, Merino E, et al. A novel class of peptide found in scorpion venom with neurodepressant effects in peripheral and central nervous system of the rat[J]. Biochimica et Biophysica Acta, 2003, 1649(1):58-67.
[8] Wang S, Jiang T Y, Ma M X, et al. Preparation and evaluation of anti-neuroexcitation peptide (ANEP) loaded N-trimethyl chitosan chloride nanoparticles for brain-targeting[J]. International Journal Pharmaceutics, 2010, 386(1/2):249-255.
[9] Zilberberg N, Gordon D, Pehate M, et al. Functional expression and genetic alteration of an alpha scorpion neurotoxin[J]. Biochemistry, 1996, 35:10215-10222.
[10] Michael T, Sharon R, Zilberberg N, et al. In vitro folding and functional analysis of an anti-insect selective scorpion depressant neurotoxin produced in Escherichia coli[J]. Protein Expression and Purification, 1997, 10:123-131.
[11] Li C, Liu W, Bossmans F, et al. Expression, renaturation and functional analysis of an excitatory insect-specific toxin from scorpion Buthus martensii Karsch[J]. Protein and Peptide Letters, 2005, 12(7):635-638.
[12] Pang S Z, Oberhaus S M, Rasmussen J L, et al. Expression of a gene encoding a scorpion peptide in yeast bacteria and plants[J]. Gene, 1992, 116:165-172
[13] 章如安, 杨晟, 邱荣德, 等. 巴斯德毕赤酵母表达体系研究及进展[J]. 微生物学通报, 2000, 27(5):371-373.
[14] 闵兆升, 郭会明, 颜旭等. 巴斯德毕赤酵母(P. pastoris)高密度发酵研究进展[J]. 生物技术通报, 2014(3):42-49.
[15] Martin-Eauclaire M F, Sogaard M, Rasmussen J L, et al. Production of active, insect-specific scorpion neurotoxin in yeast[J]. European Journal of Biochemistry, 1994, 223(2):637-645.
[16] Kjeldsen T, Pettersson A F, Hach M. Secretory Expression and Characterization of Insulin in Pichia pastoris[J]. Biotechnology and Applied Biochemistry, 1999, 29:79-86.
[17] Macauley P S, Fazenda M L, McNeil B, et al. Heterologous protein production using the Pichia pastoris expression system[J]. Yeast, 2005, 22:249-270.
[18] Cereghino G P, Cereghino J L, Ilgen C, et al. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris[J]. Current Opinion in Biotechnology, 2002, 13(4):329-332.
[19] Romanos M A, Scorer C A, Clare J J. Foreign gene expression in yeast:a review[J]. Yeast, 1992, 8:423-488.
[20] Brieley R A, Davis G R, Holtz G C. Production of insnlin-like growth factor 1 in methylotrophic yeast cells[P]. U. S. Patent, No. 5324639, 1994.
[21] 邓兵兵, 方宏清, 薛冲, 等. 人肝细胞生长因子(hdHGF)基因在毕赤酵母中的分泌表达[J]. 中国生物化学与分子生物学报, 2001, 17(5):590-594.
[22] Clare J J, Rayment F B, Ballantine S P, et al. High level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene[J]. Biotechnology, 1991, 9(5):455-460.
[23] Sreekrishna K, Nelles L, Potenz R, et al. High level expression purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast pichia pastoris[J]. Biochemistry, 1989, 28:4117-4125.
[24] Weiss H M, Haase W, Michel H, et al. Expression of functional mouse 5-HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris:pharmacological characterization and localization[J]. FEBS Letters, 1995, 377(3):451-456.
[25] 吉永华, 赵菁. 蝎抗昆虫毒素基因病毒距生物杀虫剂"任重道远"[J]. 生命科学, 1996, 8(3):37-39.