Isolation and Purification of His-CYP6J1 Fusion Protein from Aphis gossypii and Preparation of Its Polyclonal Antibody

doi:10.13560/j.cnki.biotech.bull.1985.2016-0920

Abstract

Abstract: This work aims to purify His-CYP6J1 fusion protein through affinity chromatography,and prepare and identify its polyclonal antibody. The fusion protein was purified by Ni-NTA chelating column,the purified product was detected by SDS-PAGE. Then the acquired fusion protein His-CYP6J1 was used to immune mice for preparing its polyclonal antibody against Aphis gossypii. Finally,ELISA was used to test antibody’s titer,and immunohistochemistry to identify its specificity. As results,antiserum titer was 1：200 000,the polyclonal antibody specifically bound to natural P450 CYP6J1 protein from A. gossypii,but not to the one from cotton bollworm. These results provide a foundation for studying the structure and function of a single P450 protein and its role in the insecticide- resistance of A. gossypii.

Key words: P450 CYP6J1 fusion protein, protein purification, polyclonal antibody

WANG Ya-mei WEI Yuan-jie AI Xin-yu LIU Xiao-ning. Isolation and Purification of His-CYP6J1 Fusion Protein from Aphis gossypii and Preparation of Its Polyclonal Antibody[J]. Biotechnology Bulletin, 2017, 33(5): 164-169.

References

[1] Fire A, Xu SQ, Montgomeyr MK, et al. Potent and specific genetic interference by double-strnaded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391：806-811.
[2] Kennerdell JR, Carthew RW. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway[J]. Cell, 1998, 95（7）：1017-1026.
[3] Timmons L, Fire A. Specific interference by ingested dsRNA[J]. Nature, 1998, 395（6705）：854.
[4] Stegeman JJ, Livingstone DR. Forms and functions of cytochrome P450[J]. Comparative Biochemistry & Physiology Part C Pharmacology Toxicology & Endocrinology, 1998, 121（1-3）：1-3.
[5] Brun A, Cuany A, Le Mouel TL, et al. Inducibility of the Drosophila melanogaster, cytochrome P450 gene, CYP6A2, by phenobarbital in insecticide susceptible or resistant strains[J]. Insect Biochemistry & Molecular Biology, 1996, 26（7）：697-703.
[6] 唐涛, 刘雪源, 邱立红, 等. RNA干扰及其对昆虫抗药性相关基因的沉默研究[J]. 棉花学报, 2010, 22（6）：617-624.
[7] Bernhardt R. RNA interference and its application on silencing of insecticide-resistant genes in insects[J]. Journal of Biotechnology, 2006, 124（1）：128-45.
[8] Blackman RL, Eastop VF. Aphids on the world’s crops trees：an identification and information guide[J]. Oriental Insects, 2000, 35（1）：400-400.
[9] Thorpe P, Cock PJA, Bos J. Comparative transcriptomics and proteomics of three different aphid species identifies core and diverse effector sets[J]. BMC Genomics, 2016, 17（1）：1-18.
[10] 王荫长, 韩召军. 我国农业害虫抗药性发生概况[J]. 应用昆虫学报, 1991（2）：120-121.
[11] Ahuja I, Rohloff J, Bones AM. Defence mechanisms of Brassicaceae：implications for plant-insect interactions and potential for integrated pest management. A review[J]. Agronomy for Sustainable Development, 2010, 30（2）：311-348.
[12] 龚坤元, 张桂林, 翟桂荣. 棉蚜对“1059”抗药性的测定[J]. 昆虫学报, 1964, 13（1）：1-9.
[13] Liu N, Zhu F, Xu Q, et al. Behavioral change, physiological modification, and metabolic detoxification：mechanisms of insecticide resistance[J]. Acta Entomologica Sinica, 2006, 49：671-679.
[14] Delorme R, Augé D, Béthenod MT, et al. Insecticide resistance in a strain of Aphis gossypii from Southern France[J]. Pesticide Science, 1997, 49（1）：90-96.
[15] Silver ARJ, Emden HFV, Battersby M. A biochemical mechanism of resistance to pirimicarb in two glasshouse clones of Aphis gossypii[J]. Pest Management Science, 1995, 43（1）：21-29.
[16] Moores GD, Gao X, Denholm I, et al. Characterisation of insensitive acetylcholinesterase in insecticide-resistant Cotton Aphids, Aphis gossypii glover（Homoptera：Aphididae）[J]. Pesticide Biochemistry & Physiology, 1996, 56（2）：102-110.
[17] Gong YH, Yu XR, Shang QL, et al. Oral delivery mediated RNA interference of a carboxylesterase gene results in reduced resistance to organophosphorus insecticides in the cotton Aphid, Aphis gossypii Glover[J]. PLoS One, 2014, 9（8）：e102823.
[18] 吕敏, 孙婳婳, 王丽红, 等. 植物次生物质对棉蚜谷胱甘肽S-转移酶和羧酸酯酶活性的诱导作用[J]. 中国农学通报, 2012, 28（3）：253-256.
[19] Scott JG. Cytochromes P450 and insecticide resistance[J]. Insect Biochemistry & Molecular Biology, 1999, 29（9）：757-777.
[20] Vontas J, Blass C, Koutsos AC, et al. Gene expression in insecticide resistant and susceptible Anopheles gambiae strains constitutively or after insecticide exposure[J]. Insect Molecular Biology, 2005, 14（5）：509-521.
[21] Puinean AM, Foster SP, Oliphant L, et al. Amplification of a cytochrome P450 gene is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae[J]. PLoS Genetics, 2010, 6（6）：e1000999.
[22] Kalajdzic P, Markaki M, Oehler S, et al. Imidacloprid does not induce cyp genes involved in insecticide resistance of a mutant Drosophila melanogaster line[J]. Food & Chemical Toxicology, 2013, 60（10）：355-359.
[23] 郭天凤, 史雪岩, 高希武, 等. 棉蚜吡虫啉、啶虫脒不同品系解毒酶活性测定和增效剂作用的研究[J]. 环境昆虫学报, 2014, 36（3）：388-394.
[24] Peng T, Pan Y, Yang C, et al. Over-expression of CYP6A2, is associated with spirotetramat resistance and cross-resistance in the resistant strain of Aphis gossypii, Glover[J]. Pesticide Biochemistry & Physiology, 2015, 126：64-69.
[25] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 2015, 72（1-2）：248-254.
[26] GRTB. Immunology—A short course（2nd edition）：Editors. E Benjamini and S Leskowitz[M]. New York：Wiley-Liss, 1991：459pp.
[27] 黄丽娜, 程婷婷, 王新绘, 等. 棉铃虫β-actin基因的克隆、表达及多克隆抗体制备[J]. 生物技术通报, 2015, 31（12）：138-145.
[28] 魏原杰, 张雷, 程婷婷, 等. 棉铃虫细胞色素P450 CYP6B6多克隆抗体的制备[J]. 生命科学研究, 2015（6）：497-500.
[29] Chang GD, Li ZL, Qin JY, et al. Optimization of fermentation of recombinant human Endostatin（rh-Endostatin）expression in Escherichia coli[J]. Chinese Journal of Biotechnology, 2005, 21（4）：662-666.
[30] 李东江, 郑颖, 叶锋平, 等. 蛇毒类凝血酶基因大肠杆菌和酵母细胞表达条件的优化[J]. 西南国防医药, 2010, 20（6）：670-672.
[31] 于殿宇, 罗淑年, 王瑾, 等. 海藻酸钠-明胶固定化磷脂酶的研究[J]. 食品工业, 2008（3）：9-11.
[32] 刘玉素. 棉蚜腹管的组织构造和几个关于腹管功能的试验[J]. 动物学报, 1956, 8（1）：17-28.