Cloning of Antibacterial Peptide Gene and Auto-induction Expressing of Recombinant Proteins from Bombyx mori

doi:10.7668/hbnxb.2018.01.006

Abstract

Abstract: Antibacterial peptide is a kind of small molecular compound with antibacterial activity in organisms,especially in insect cells that resist the infection of foreign microbe,to research cloning of antibacterial peptide genes and expressing and purification of recombinant proteins from Bombyx mori. Four pairs of special primers were designed,and four genes BmCecropin,BmCecropinB6,BmCecropinD and Bmmoricin were amplified by RT-PCR from total RNA extracted from midgut from silkworm,then four recombinant proteins were expressed and purified by auto-induction and Ni-NTA affinity chromatography and ultrafiltration purification.The results showed that four antibacterial peptide genes were 198,108,105,129 bp in sizes,respectively.Further SDS-PAGE electrophoresis to detect four recombinant antibacterial peptide,results showed that four recombinant antibacterial peptide by auto-induction expressing were 24,21,20,22 ku in sizes,respectively.The four purified antibacterial peptides constructed a basis on subsequent antibacterial test and further application on bacteriostatic agent,cosmetics and preservatives.

Key words: Bombyx mori, Antibacterial peptide, Recombinant protein, Auto-induction expressing, Purified

CLC Number:

XIE Kun, WANG Jing, WANG Lixian, ZHU Lingming, YIN Jianhua, YE Qingxia, SUN Yan. Cloning of Antibacterial Peptide Gene and Auto-induction Expressing of Recombinant Proteins from Bombyx mori[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(1): 33-38. doi: 10.7668/hbnxb.2018.01.006.

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References

[1] Baman H G, Nilsson I, Rasmuson B. Inducible antibactercial denfense system in Drosophila[J]. Nature,1972,237:232-235.
[2] Lakshmaiah N J, Chen J Y. Antimicrobial peptides:Possible anti-infective agents[J]. Peptides, 2015, 72(10):88-94.
[3] Sancho-Vaello E, Zeth K. Antimicrobial peptides:has their time arrived?[J]. Future Microbiol, 2015, 10(7):1103-1106.
[4] Ezzati-Tabrizi R, Farrokhi N, Talaei-Hassanloui R, et al. Insect inducible antimicrobial peptides and their applications[J]. Curr Protein Pept Sci, 2013, 14(8):698-710.
[5] Cheng T, Zhao P, Liu C, et al. Structures, regulatory regions, and inductive expression patterns of antimicrobial peptide gens in the silkworm Bombyx mori[J]. Genomies, 2006, 87(3):356-365.
[6] 孙伟,沈以红,向仲杯,等.家蚕抗菌肽基因研究进展[J].蚕业科学,2009,35(1):196-203.
[7] Yang W,Cheng T,Ye M,et al.Functional divergence among silkworm antimicrobial peptide paralogs by the activities of recombinant proteins and the induced expression profiles[J].PLoS One,2011,6(3):e18109.
[8] Studier F W.Protein production by auto-induction in high density shaking cultures[J].Protein Expression and Purification,2005,41(1):207-234.
[9] Studier F W. Stable expression clones and auto-induction for protein production in E.coli[J]. Methods Mol Biol, 2014, 1091:17-32.
[10] EL-Baky N A, Linjawi M H, Redwan E M. Auto-induction expression of human consensus interferon-alpha in Escherichia coli[J]. BMC Biotechnol, 2015,15:14.
[11] J萨姆布鲁克,拉塞尔.分子克隆实验指南[M].4版.黄培堂,等译.北京:科学出版社,2016.
[12] Taniguchi M,Ochiai A,Takahashi K,et al.Antimicrobial activity against Porphyromonas gingivalis and mechanism of action of the cationic octadecapeptide AmyI-1-18 and its amino acid-substituted analogs[J].Journal of Bioscience and Bioengineering,2016,122(6):652-659.
[13] Potturu M,Prabhakaran P A,Neetu O N,et al.Cathelicidin expression and role in oral health and diseases:A short review[J].Tropical Journal of Medical Research,2017,17(2):69-75.
[14] Benergossi J,Calixto G,Fonseca-Santos B,et al.Highlights in peptide nanoparticle carriers intended to oral diseases[J].Current Topics in Medicinal Chemistry,2015,15(4):345-355.
[15] Borovaya A,Dombrowski Y,Zwicker S,et al.Isotretinoin therapy changes the expression of antimicrobial peptides in acne vulgaris[J].Archives of Dermatological Research,2014,306(8):689-700.
[16] 王志涛,王俊刚,申红.昆虫抗菌肽研究进展[J].新疆农垦科技,2015,6:36-38.
[17] 郝思怡,姚远航,郭土敬,等.金环蛇抗菌肽在毕赤酵母中的组成型表达[J].生物技术,2015,25(2):124-127.
[18] Yang S,Yang F,Wei N,et al.A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1[J].Nature Communications,2015,6(3):8297.
[19] 李娟,吴永胜,杨雪. 蚯蚓抗菌肽研究进展[J]. 安徽农业科学, 2016,44(3):62-63.
[20] 张彦明,刘美辰, 孙雨萱. 金黄色葡萄球菌蛋白A在大肠杆菌中的自诱导表达以及单克隆抗体的制备[J]. 粮食科技与经济, 2016, 41(4):31-35.
[21] 王昭,王席,杨威, 等. 金属硫蛋白基因MT1A 在大肠杆菌中的自诱导表达条件优化及其抗镉性[J]. 河南农业科学, 2016, 45(4):66-70.
[22] 彭冬,杨威王昭, 等. GST-SUMO-MT融合蛋白在大肠杆菌中的自诱导表达[J]. 生物技术通报, 2016, 32(2):219-224.
[23] 陈可,代娟,甘春扬, 等. 通过自诱导方法对乙型肝炎病毒聚合酶TP区进行可溶性表达及鉴定[J]. 中国生物制品学杂志, 2017, 30(1):25-33.
[24] 章玉萍,牛晓利,曹玉桥,等.家蚕抗菌肽Cecropin D基因的原核表达,纯化及抑菌活性测定[J].中国蚕业,2012,32(4):20-23.
[25] 张学敏,金莉莉,王铮,等.抗菌肽在大肠杆菌中的融合表达[J].生物工程学报,2014,30(8):1172-1181.
[26] Li Y.Recombinant production of antimicrobial peptides in Escherichia coli:a review[J].Protein Expression and Purification,2011,80(2):260-267.
[27] Aleinein R A,Hamoud R,Schäfer H,et al.Molecular cloning and expression of ranalexin,a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities[J].Applied Microbiology and Biotechnology,2013,97(8):3535-3543.

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