Eukaryotic Expression of Antibacterial Peptide Domesticin from Musca domestica in Pichia Pastoris and Antibacterial Activity Identification

doi:10.13560/j.cnki.biotech.bull.1985.2018-0611

Abstract

Abstract: This work aims to express the Musca domestica antibacterial peptide Domesticin gene domesticin in Pichia pastoris and determine the antibacterial activity of the expressed product. The cloned domesticin gene fragment was ligated to pPIC9K vector to construct a recombinant expression vector pPIC9K-domesticin,which was transformed into competent Pichia pastoris KM71. Recombinant fusion protein Domesticin was induced with methanol,the expressed product was identified by Tricine-SDS-PAGE and Western Blot,and antibacterial activity was determined by minimal inhibitory concentration. The results indicated that antibacterial peptide Domesticin was successfully expressed in P. pastoris,and Domesticin demonstrated antibacterial activity against many kinds of bacteria. Domesticin is a novel broad spectrum antibacterial peptide that has antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria,which has the capacity to be a new generation antimicrobial agent.

Key words: Musca domestica, antibacterial peptide, Pichia pastoris, eukaryotic expression, antibacterial activity

LIU Jin-lan, YANG Xue, LI Shuang-shuang, ZHANG Yu-ming, LIU Feng-song, TANG Ting, LI Hong-quan. Eukaryotic Expression of Antibacterial Peptide Domesticin from Musca domestica in Pichia Pastoris and Antibacterial Activity Identification[J]. Biotechnology Bulletin, 2019, 35(2): 109-115.

References

[1] Brogden KA.Antimicrobial peptides:pore formers or metabolic inhibitors in bacteria[J]. Nature Reviews Microbiology, 2005, 3
(3):238-250.
[2] Hancock RE, Chapple DS.Peptide antibiotics[J]. Lancet, 1999, 43(9049):418-422.
[3] Oren Z, Shai Y.Mode of action of linear amphipathic alpha-helical antimicrobial peptides[J]. Peptide Science, 1998, 47(6):451-463.
[4] Yount NY, Bayer AS, Xiong, YQ, et al.Advances in antimicrobial peptide immunobiology[J]. Peptide Science, 2010, 84(5):435-458.
[5] English BK, Gaur AH.The use and abuse of antibiotics and the development of antibiotic resistance[J]. Hot Topics in Infection and Immunity in Children VI, 2010:659:73-82.
[6] 尹昆仑, 王嘉榕, 孙红宾. 抗菌肽的研究进展及应用前景[J].中国生化药物杂志, 2015, 35(5):181-185.
[7] Hamamoto K, Kida Y, Zhang Y, et al.Antimicrobial activity and stability to proteolysis of small liner cationic peptides with D-amino acid substitutions[J]. Microbiology and Immunology, 2013, 46(11):741-749.
[8] Hoffmann JA.The immune response of Drosophila[J]. Nature, 2003, 426(6962):33-38.
[9] Tanaka H, Ishibashi J, Fujita K, et al.A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori[J]. Insect Biochemistry and Molecular Biology, 2008, 38(12):1087-1110.
[10] Graveley BR, Kaur A, Gunning D, et al.The organization and evolution of the dipteran and hymenopteran Down syndrome cell adhesion molecule(Dscam)genes[J]. RNA, 2004, 10(10):1499-1506.
[11] Kocks C, Cho JH, Nehme N, et al.Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila[J]. Cell, 2005, 123(2):335-346.
[12] 柳峰松, 孙玲玲, 唐婷, 等. 家蝇抗菌肽Attacin-2基因的克隆序列分析和诱导表达[J]. 昆虫学报, 2011, 54(1):27-33.
[13] Lu X, Shen J, Jin X, et al.Bactericidal activity of Musca domestica cecropin(Mdc)on multidrug-resistant clinical isolate of Escherichia coli[J]. Appl Microbiol Biotechnol, 2012, 95(4):939-945.
[14] 柳峰松, 王丽娜, 唐婷等. 家蝇抗菌肽Diptericin基因的克隆与分析[J]. 昆虫学报, 2009, 52(10):1078-1082.
[15] Wang JX, Zhao XF, Liang YL, et al.Molecular characterization and expression of the antimicrobial peptide defensin from the housely(Musca domestica)[J]. Cellular and Molecular Life Sciences:CMLS, 2006, 63(24):3072-3082.
[16] Pei ZH, Sun XN, Tang Y, et al.Cloning, expression, and purification of a new antimicrobial peptide gene from Musca domestica larva[J]. Gene, 2014, 549(1):41-45.
[17] 杨雪, 唐婷, 王一丽, 等. 家蝇新型抗菌肽Muscin的基因克隆、表达模式及抑菌活性[J]. 昆虫学报, 2015, 58(6):617-624.
[18] 杨雪. 两种新型抗菌肽基因muscin和domesticin的鉴定[D]. 保定:河北大学, 2015:28.
[19] 冉琳, 何钢, 刘嵬, 等. 大头金蝇幼虫抗菌肽提取纯化及抑菌活性研究[J]. 食品科技, 提取物与应用, 2017, 42(10):248-252.
[20] 陆婕, 钟雅, 柳林, 等. 家蝇蛆抗菌肽提取工艺研究[J]. 昆虫学报, 2007, 50(2):106-112.
[21] Hao G, Shi YH, Tang YL, et al.Design and analysis of structure-activity relationship of novel antimicrobial peptides derived from the conserved sequence of cecropin[J]. Journal of peptide science, 2010, 14(3):290-298.
[22] Xie J, Gou Y, Zhao Q, et al.Antimicrobial activities and membrane-active mechanism of CPF-C1 against multidrug-resistant bacteria, a novel antimicrobial peptide derived from skin secretions of the tetraploid frog Xenopus clivii[J]. Journal of Peptide Science, 2015, 20(11):876-884.
[23] 凌霄. 抗菌肽cgMolluscidin和PaDef的重组表达、纯化及活性研究[D]. 天津:天津科技大学食品工程专业, 2016:33-34.
[24] Macauley-Patrick S, Fazenda ML, McNeil B, et al. Heterologous protein production using the Pichia pastoris expression system[J]. Yeast, 2010, 22(4):249-270.
[25] Tabarraei A, Kalhor HR, Armanmehr S.Production of a soluble and functional recombinant apolipoprotein in the Pichia pastoris expression system[J]. Protein Expression and Purification, 2016, 121(5):157-162.
[26] Xu F, Meng K, Wang Y R, et al.Eukaryotic expression and antimicrobial spectrum determination of the peptide tachyplesin II[J]. Protein Expression and Purification, 2008, 58(2):175-183.
[27] Luiz D, Almeida J, et al.Heterologous expression of abaecin peptide from Apis mellifera in Pichia pastoris[J]. Microbial Cell Factories, 2017, 16(5):76.
[28] 蔡晶晶, 杨明, 蔡灵, 等. 黑鲷抗菌肽hepcidin在毕赤酵母中的表达及其抗菌活性[J]. 厦门大学学报:自然科学版, 2009, 48(5):738-743.
[29] Chen XM, Li J, Sun HL, et al.High-level heterologous production and Functional Secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide Apidaecin[J]. Scientific Reports. 2017, 7:14543.
[30] Li WX, Shi XT, Wei G, et al.Recombinant expression and biological characterization of the antimicrobial peptide fowlicidin-2 in Pichia pastoris[J]. Experiment and Therapeutic Medicine. 2016, 12(4):2324-2330.