东北林蛙皮肤抗菌肽dybowskin-1ST的表达、纯化及抑菌活性分析

doi:10.3864/j.issn.0578-1752.2011.15.020

Abstract

Abstract: 【Objective】 The aim of this study is to express Rana dybowskii antimicrobial peptide dybowskin-1ST via Pichia pastoris system and to analyze its antibiotic activity.【Method】The total RNA of Rana dybowskii skin was extracted by TRIzol method. The antimicrobial peptide dybowskin-1ST DNA were obtained by RT-PCR and cloned into expression vector pPIC9K, and recombinant pPIC9K-1ST was obtained. Then, the pPIC9K-1ST was electrotransformed into Pichia pastoris GS115, and the recombinant strain expressing dybowskin-1ST was generated. The recombinant strain was induced by the methanol and the interest protein was expressed via SDS-PAGE analysis. The interest protein was purified with Ni-agarose and its antimicrobial activity was detected using Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella enteritidis, Pseudomonas aeruginosa and Klebsiella pneumoniae.【Result】The recombinant Pichia Pastoris system expressing Rana dybowskii antimicrobial peptide dybowskin-1ST was well-constructed and the peptide dybowskin-1ST showed significant antimicrobial activity on gram-positive bacteria and gram-negative bacteria.【Conclusion】Rana dybowskii antimicrobial peptide dybowskin-1ST has a broad range of antimicrobial activity and a number of potential applications.

Key words: Rana dybowskii, antimicrobial peptide, Pichia pastoris, antibacterial activity

GUO Na, XIAO Xiang-Hong, XU Yi-Gang, CHAI Long-Hui, ZHANG Jing-Yu. Expression, Purification and Antibacterial Activity Analysis of Rana dybowskii Antimicrobial Peptide Dybowskin-1ST[J].Scientia Agricultura Sinica, 2011, 44(15): 3246-3251.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2011.15.020

https://www.chinaagrisci.com/EN/Y2011/V44/I15/3246

References

[1]Hancock R E W. Cationic peptide: effectors in innate immunity and novel antimicrobials. The Lancet Infectious Disease, 2001, 1(3): 156-164.

[2]Koczulla A R, Robert B. Antimicrobial peptides: current status and therapeutic potential. Drugs, 2003, 63(4): 389-406.

[3]Wu Z B, Powell R, Lu W Y. Productive folding of human neutrophil α-defensins in vitro without the pro-peptide. Journal of the American Chemical Society, 2003, 125(9): 2402-2403.

[4]Boulanger N, Ehret-Sabatier L, Brun R, Zachary D, Bulet P, Imler J L. Immune response of Drosophila melanogaster to infection with the flagellate parasite Crithidia spp. Insect Biochemistry and Molecular Biology, 2001, 31: 129-137.

[5]Zasloff M. Antimicrobial peptides of multicellular organisms. Nature, 2002, 415: 389-395.

[6]申屠德君. 特定细菌刺激下东北林蛙皮肤抗菌肽基因克隆及其表达[D]. 黑龙江: 东北林业大学, 2009.

Shentu D J. Cloning and differential expression of antibacterial peptide genes from the skin of Rana dybowskii after bacterial stimulation[D]. Heilongjiang: Northeast Forestry University, 2009. (in Chinese)

[7]Csordas A, Michl H. Isolation and structure of a haemolytic polypeptide from the defensive secretion of European Bombina species. Monatshefte Chemistry, 1970, 101: 182-189.

[8]Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proceedings of the National Academy of Sciences of the USA, 1987, 84: 5449-5453.

[9]Soballe P W, Maloy W L, Myrga M L, Jacob L S, Herlyn M. Experimental local therapy of human melanoma with lytic magainin peptides. International Journal of Cancer, 1995, 60: 280-284.

[10]杨阳, 孙红瑜, 金光耀, 杨娇. 抗菌肽的作用机理及应用前景. 中国林副特产, 2010, 2(104): 103-104.

Yang Y, Sun H Y, Jin G Y, Yang J. The role of the mechanism and prospect of antimicrobial peptides. Forest by-Product and Speciality in China, 2010, 2(104): 103-104. (in Chinese)

[11]黄现清, 高晓平, 赵改名, 李苗云, 柳艳霞, 张秋会, 孙灵霞, 袁勇军. 抗菌肽抑菌机制研究进展. 生物学杂志, 2010, 27(2): 62-66.

Huang X Q, Gao X P, Zhao G M, Li M Y, Liu Y X, Zhang Q H, Sun L X, Yuan Y J. Review of bacteriostasis mechanisms of antimicrobial peptides. Journal of Biology, 2010, 27(2): 62-66. (in Chinese)

[12]Finlay B B, Hancock R E W. Opinion: can innate immunity be enhanced to treat microbial infections. Nature Reviews Microbiology, 2004, 2: 497-504.

[13]Hang H F, Ye X B, Guo M J, Chu J, Zhuang Y P, Zhang M, Zhang S L. A simple fermentation strategy for high-level production of recombinant phytase by Pichia pastoris using glucose as the growth substrate. Enzyme and Microbial Technology, 2009, 44: 185-188.

[14]娄瑞娟, 罗利龙, 张霞, 张瑞刚, 宋水山. 巴斯德毕赤酵母表达系统的研究进展和前景展望. 生物学杂志, 2010, 27(5): 73-76.

Lou R J, Luo L L, Zhang X, Zhang R G, Song S S. Research progress and prospects on Pichia pastoris. Journal of Biology, 2010, 27(5): 73-76. (in Chinese)

[15]Çelik E, Çalik P, Oliver S G. A structured kinetic model for recombinant protein production by Mut+ strain of Pichia pastoris. Chemical Engineering Science, 2009, 64: 5028-5035.

[16]Straus S K, Hancock R E W. Mode of action of the new antibiotic for gram-positive pathogens daptomycin: comparison with cationic antimicrobial peptides and lipopeptides. Biochimica et Biophysica Acta, 2006, 1758(9): 1215-1223.

[17]Urbán E, Nagy E, Pál T, Agnes Á, Conlon J M. Activities of four frog

skin-derived antimicrobial peptides (temporin-1DRa, temporin-1Va and the melittin-related peptides AR-23 and RV-23) against anaerobic bacteria. International Journal of Antimicrobial Agents, 2007, 29: 317-321.

[18]Hawkey P M. The origins and molecular basis of antibiotic resistance. British Medical Journal, 1998, 317(7159): 657-660.

[19]Conlon J M, Kolodziejek J, Nowotny N. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochimica et Biophysica Acta, 2004, 1696: 1-14.

[20]UniProtKB/SWISS-PROT at EBI. http://www.expasy.org/sprot/relnotes/ relstat.html.

[21]Lee D G, Park Y, Kim H N, Kim H K, Kim P Il, Choi B H, Hahm K S. Antifungal Mechanism of an antimicrobial peptide, HP (2-20), derived from N-Terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans. Biochemical and Biophysical Research Communications, 2002, 291(4): 1006-1013.

[22]Gazit E, Lee W J, Brey P T, Shai Y. Mode of action of the antibacterial cecropin B2: a spectrofluorometric study. Biochemistry, 1994, 33(35): 10681-10692.

[23]Reddy K V R, Yedery R D, Aranha C. Antimicrobial peptides: premises and promises. International Journal of Antimicrobial Agents, 2004, 24: 536-547.

[24]金莉莉. 东北林蛙皮抗菌肽及其生物学特性[D]. 沈阳: 中国医科大学, 2009.

Jin L L. Identification and characterization of antimicrobial peptides from the skin secretion of a Chinese Frog (Rana dybowskii)[D]. Shenyang: Chinese Medical University, 2009. (in Chinese)

Related Articles 11

[1]	ZHANG Qi,DUAN Yu,SU Yue,JIANG QiQi,WANG ChunQing,BIN Yu,SONG Zhen. Construction and Application of Expression Vector Based on Citrus Leaf Blotch Virus [J]. Scientia Agricultura Sinica, 2022, 55(22): 4398-4407.
[2]	CHEN WenFeng,WANG HongFang,LIU ZhenGuo,ZHANG WeiXing,CHI XuePeng,XU BaoHua. Recombinant Expression and Antimicrobial Activity of Apidaecin in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2019, 52(4): 767-776.
[3]	ZHANG Ke-Xin, ZHANG Ming-Yue, XIN Sheng-Nan, HAN Zong-Xi, SHAO Yu-Hao, LIU Sheng-Wang, MA De-Ying. Isolation, Characterization and Mechanism of Anti-viral Activity of Duck Avian Beta-defensin 16 [J]. Scientia Agricultura Sinica, 2012, 45(18): 3873-3882.
[4]	HE Yong-Sheng, CHEN Shu-Chi, WANG Hong, HAN Shuang-Yan, YANG Qiong, LIU Xi-Xia, DONG Jie-Xian, YANG Wu-Ying, SUN Yuan-Ming. Expression of Bombyx mori Acetylcholinesterase Gene bmace in Pichia pastoris GS115 and Analysis of Its Bioactivity [J]. Scientia Agricultura Sinica, 2012, 45(11): 2189-2198.
[5]	LI Rong-rong,HE Zhen-quan,BAO En-dong,CHEN Pu-yan . Expression of the Chicken Antibacterial Peptide Fowlicidin-3 in Pichia Pastoris and Identification of Biological Activity#br# [J]. Scientia Agricultura Sinica, 2010, 43(21): 4529-4535 .
[6]	WU Jian-ming,WANG Chang-fa,HE Hong-bin,HU Gui-xue,YANG Hong-jun,YANG Shao-hua,GAO Yun-dong,ZHONG Ji-feng . Cloning and Expression of cDNA of Bovine Neutrophil β-defensin12 from Holstein Cow and Its Antibacterial Activity [J]. Scientia Agricultura Sinica, 2010, 43(2): 396-403 .
[7]	. Cloning and Expression of a Neutral Protein Gene BS2 from Bacillus subtilis B111 in Pichia pastoris [J]. Scientia Agricultura Sinica, 2009, 42(3): 876-883 .
[8]	. Secreted Expression of the Combinant Defensin alfAFP (M. sativa) in Pichia pastoris and Its Antimicrobial Activity Against Rice Pathogens in Vitro [J]. Scientia Agricultura Sinica, 2009, 42(3): 869-875 .
[9]	. Expression of Pseudomonas aeruginosa Alginate Lyase Gene(algL) in Pichia pastoris and Primary Analysis of Enzymic Properties [J]. Scientia Agricultura Sinica, 2008, 41(4): 1192-1198 .
[10]	. Secretive expression of insect antifungal peptide genes in Pichia pastoris and activity detection of expressive products [J]. Scientia Agricultura Sinica, 2007, 40(4): 842-849 .
[11]	. Expression and characterization of Porcine Epidermal Growth Factor in Pichia Pastoris [J]. Scientia Agricultura Sinica, 2007, 40(11): 2593-2599 .

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Expression, Purification and Antibacterial Activity Analysis of Rana dybowskii Antimicrobial Peptide Dybowskin-1ST

PDF

Cited