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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (6): 9-16    DOI: 10.13523/j.cb.20180602
    
Expression of Fusion Antibacterial Peptide in Recombinant Pichia pastoris and Its Bioactivity In Vitro
Jian-xue TANG,Yong-le XIAO,Jun-jie PENG,Shi-ji ZHAO,Xiao-ping WAN,Rong GAO()
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, China
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Abstract  

Objective:To express fusion antimicrobial peptide in Pichia pastoris SMD1168 and determine its in vitro bioactivity.Methods:The constructed fusion anti-peptide RHKJT gene fragment was cloned from the recombinant pVAX1-RHKJT vector previously constructed in laboratory. The RHKJT gene fragment was inserted into plasmid pGAPZaA, then verified by PCR and sequenced to construct recombinant pGAPZα-RHKJT vector. The linearized pGAPZα-RHKJT was electroporated into Pichia pastoris SMD1168 to obtain the recombinant yeast SMDpG-RHKJT. The recombinant Pichia pastoris SMDpG-RHKJT was fermented and confirmed by PCR and RT-PCR. Fermentation supernatants were collected for in vitro bioactivity assay.Results:The recombinant yeast SMDpG-RHKJT was successfully obtained, and the recombinant yeast’s fermentation supernatant had significant inhibition effects on Escherichia coli, Salmonella, Staphylococcus aureus and Streptococcus pneumonia.Conclusion:The recombinant antimicrobial peptide expressed by recombinant yeast has marked antibacterial activity and would facilitate the development of novel antibacterial additive feed later.

Key words： Fusion antibacterial peptide      Gene expression      Recombinant Pichia pastoris bacteriostasis      Antibacterial bioactivity     
Received: 02 January 2018      Published: 06 July 2018
ZTFLH:  Q786  
Corresponding Authors: Rong GAO     E-mail: gaorong96@163.com
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Jian-xue TANG
Yong-le XIAO
Jun-jie PENG
Shi-ji ZHAO
Xiao-ping WAN
Rong GAO
Cite this article:

Jian-xue TANG,Yong-le XIAO,Jun-jie PENG,Shi-ji ZHAO,Xiao-ping WAN,Rong GAO. Expression of Fusion Antibacterial Peptide in Recombinant Pichia pastoris and Its Bioactivity In Vitro. China Biotechnology, 2018, 38(6): 9-16.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180602     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I6/9

片段 引物名称 序列(5'- 3') 长度
(bp)
FD F-FD GAAGCTGAATTCATGGGAATCATAAACACATTACAGA 37
R-FD GTCCCCGCATGTTAGAAGACTTCCCCTGCCCTCTCCGCTTCCTGGCTTTTTGCAGCATTTT 61
FD+2A-α F-FD-2A-α AATTGGAACCTGCGGTCTCCCTGGAACAAAATGCTGCAAAAAGCCAGGAAGCGGAGAGGGCA 62
R-FD-2A-α ATGATTTCTTACTCCTTGCATAGCTTCAGCCTCTCTTTTCTC 42
BNBD3/HNP3 F-BNBD3/HNP3 GAGGCTGAAGCTATGCAAGGAGTAAGAAATCATG 34
R-BNBD3/HNP3 TTAGAAGACTTCCCCTGCCCTCTCCGCTTCCGCAGCAGAATGCCCAGAGTC 51
BNBD3/
HNP3+2-α		 F-2A-α(1) CGCTATGGCACCTGCATCTACCAGGGAAGACTCTGGGCATTCTGCTGCGGAAGCGGAGAGGGCA 64
R-2A-α(1) AGTAGATTTTTGAGCTTTTTACCCGCGAGTTTAGAGAATATTCCCATAGCTTCAGCCTCTCTTT
TCTC		 68
ECD F-ECD GCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTATGGG
AATATTCTCTAAACTCGC		 80
R-ECD GTCCCCGCATGTTAGAAGACTTCCCCTGCCCTCTCCGCTTCCGTATTTACGCAGAGAGAACAGCA 65
ECD+2A+α F-ECD-2A-α GGTAAGCGCCTCTTCAAGAAGCTGCTGTTCTCTCTGCGTAAATACGGAAGCGGAGAGGGCA 61
R-ECD-2A-α CGCCACCTAGCAGAGTTTTAATACGCTTTAGTAAGCCCATAGCTTCAGCCTCTCTTTTCTC 61
AJI F-AJI GAGAGGCTGAAGCTATGGGCTTACTAAAGCGTATTAA 37
R-AJI CTAGTCTAGACTAAAAAAGCTTCTTTATTTTCCAGA 36
Table 1 Primers sequence
菌株 卡那霉素(μg/ml) 多粘菌素B
(μg/ml)
大肠杆菌标准菌 1、2、4
大肠杆菌耐药菌 2、4、8
沙门氏菌标准菌 0.5、1、2
金黄色葡萄球菌标准菌 0.5、1、2
金黄色葡萄球菌耐药菌 512、1024、2048
肺炎链球菌标准菌 2、4、8
Table 2 Antibiotic gradient concentrations for six bacterial strains
Fig.1 Electrophoregram of fused antimicrobial peptide fragments obtained by PCR amplification (1% agarose gel) Lane M:Trans 2K plus DNA marker; Lane 1:Fusion antibacterial peptide gene amplification product
Fig.2 Recombinant plasmid pGAPZαA-RHKJT PCR amplification products electrophoresis(1% agarose gel) Lane M:Trans 2K DNA marker; Lane 1~3:pGAPZαA bacterial liquid PCR target band 1 866bp
Fig.3 PCR identification of recombinant yeast (1% agarose gel) Lane M:Trans 2K plus DNA marker; Lane 1:SMDpG-RHKJT bacterial PCR target band
Fig.4 RT-PCR electrophoresis detection of recombinant yeast transcription (1% agarose gel) Lane M:Trans 2K plus DNA marker; Lane 1:SMDpG-RHKJT gene amplification
Fig.5 Bacteriostasis of standard E.coli * Values with different superscripts letters differ significantly(P<0.05) and vice visa; the followings are the same as here
Fig.6 Inhibition test of drug resistant E.coli
Fig.7 Bacteriostasis of standard Streptococcus pneumoniae bacteria
Fig.8 Bacteriostasis of standard Staphylococcus aureus bacteria
Fig.9 Bacteriostasis of resistant Staphylococcus aureus bacteria
Fig.10 Bacteriostasis of Salmonella bacteria
[1]   乔玮, 郝华, 彭会 , 等. 抗菌肽作为饲料添加剂的研究进展. 生物技术通报, 2014(10):43-48.
[1]   Qiao W, Hao H, Peng H , et al. Progress of antimicrobial peptides as feed additive. Biotechnology Bulletin, 2014(10):43-48.
[2]   Kaufmann B B, Hung D T . The fast track to multidrug resistance. Molecular Cell, 2010,37(3):297-298.
doi: 10.1016/j.molcel.2010.01.027 pmid: 20159549
[3]   Hs L . Alternatives to antibiotics: a symposium on the challenges and solutions for animal production. Animal Health Research Reviews, 2013,14(1):78-87.
doi: 10.1017/S1466252313000030
[4]   Sierra J M, Fusté E, Rabanal F , et al. An overview of antimicrobial peptides and the latest advances in their development. Expert Opinion on Biological Therapy, 2017,17(6).
doi: 10.1080/14712598.2017.1315402 pmid: 28368216
[5]   Peschel A, Sahl H G . The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nature Reviews Microbiology, 2006,4(7):529-536.
doi: 10.1016/j.scriptamat.2005.11.004 pmid: 202020202020202020202020
[6]   Otto M . Colonization of the skin and antimicrobial peptides. Expert Review of Dermatology, 2010,5(2):183-195.
doi: 10.1586/edm.10.6
[7]   燕晓翠, 杨春蕾, 姚大为 , 等. 抗菌肽的国内外研究进展. 天津农业科学, 2017,23(5):35-41.
[7]   Yan X C, Yang C L, Yao D W , et al. Research progress on domestic and abroad of antibacterial peptides. Tianjin Agricultural Sciences, 2017,23(5):35-41.
[8]   Zasloff M . Antimicrobial peptides of multicellular organisms. Nature, 2002,415(6870):389-395.
doi: 10.1038/415389a pmid: 11807545
[9]   陈晓平, 贾春兰, 马吉霞 . 杂合抗菌肽Sα-Jp基因真核表达载体的构建. 食品与发酵科技, 2014(2):9-12.
doi: 10.3969/j.issn.1674-506X.2014.02-003
[9]   Chen X P, Jia C L, Ma J X . Construction of eukaryotic expression vector for antibacterial peptide Sα-Jp gene. Food and Fermentation Technology, 2014(2):9-12.
doi: 10.3969/j.issn.1674-506X.2014.02-003
[10]   Servettaz A, Kavian N, Nicco C , et al. Controlling the release of peptide antimicrobial agents from surfaces. Biomaterials, 2010,31(8):2348-2357.
doi: 10.1016/j.biomaterials.2009.11.082 pmid: 20004967
[11]   Da C J, Cova M, Ferreira R , et al. Antimicrobial peptides: an alternative for innovative medicines?. Applied Microbiology & Biotechnology, 2015,99(5):2023-2040.
doi: 10.1007/s00253-015-6375-x pmid: 25586583
[12]   彭梅, 孙茂盛 . 重组抗菌肽基因克隆及其在Pichia pastoris中的表达及鉴定. 中国生物工程杂志, 2008(s1):27-31.
[12]   Peng M, Sun M S . Cloning and expression of antibaterial peptide gene in Pichia pastoris and identifying of activity. China Biotechnology, 2008(s1):27-31.
[13]   杨平, 袁奕豪, 杨晓莉 , 等. 抗菌肽高效表达及生产优化研究进展. 生物技术通报, 2016,32(3):24-30.
doi: 10.13560/j.cnki.biotech.bull.1985.2016.03.005
[13]   Yang P, Yuan Y H, Yang X L , et al. Research progress of efficient expression and optimization of production of antibacterial peptide. Biotechnology Bulletin, 2016,32(3):24-30.
doi: 10.13560/j.cnki.biotech.bull.1985.2016.03.005
[14]   Fink J, Boman A, Boman H G , et al. Design, synthesis and antibacterial activity of cecropin-like model peptides. International Journal of Peptide & Protein Research, 1989,33(6):412-421.
doi: 10.1111/j.1399-3011.1989.tb00217.x pmid: 2777475
[15]   武如娟, 张日俊 . 杂合抗菌肽设计及生物学活性的研究进展. 中国生物工程杂志, 2013,33(9):94-100.
[15]   Wu R J, Zhang R J . The progress of hybrid peptides on design and biological activity. China Biotechnology, 2013,33(9):94-100.
[16]   Lu X M, Jin X B, Zhu J Y , et al. Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli. Applied Microbiology & Biotechnology, 2010,87(6):2169-2176.
doi: 10.1007/s00253-010-2606-3 pmid: 20499232
[17]   Shin J R, Lim K J, Kim D J , et al. Display of multimeric antimicrobial peptides on the Escherichia coli cell surface and its application as whole-cell antibiotics. Plos One, 2013,8(3):e58997.
doi: 10.1371/journal.pone.0058997 pmid: 3597565
[18]   王秀青, 张素芳, 曹瑞兵 , 等. 抗菌肽天蚕素B基因及其串联体在毕赤酵母中的表达. 南京农业大学学报, 2007,30(3):120-123.
[18]   Wang X Q, Zhang S F, Cao R B , et al. Antibacterial peptide Cecropin B and its tandem gene expressed in Pichia pastoris. Journal of Nanjing Agricultral University, 2007,30(3):120-123.
[19]   Andreu D, Ubach J, Boman A , et al. Shortened cecropin A-melittin hybrids. Significant size reduction retains potent antibiotic activity. Febs Letters, 1992,296(2):190-194.
doi: 10.1016/0014-5793(92)80377-S
[20]   张素芳, 曹瑞兵, 贾赟 , 等. 杂合抗菌肽CecA-mil的改造及在毕赤酵母中的分泌表达. 微生物学报, 2005,45(2):218-222.
doi: 10.3321/j.issn:0001-6209.2005.02.013
[20]   Zhang S F, Cao R B, Jia Y , et al. Modification of hybrid antimicrobial peptide CecA-mil gene and its over-secretion expression in Pichia pastoris. Acta Microbiologica Sinica, 2005,45(2):218-222.
doi: 10.3321/j.issn:0001-6209.2005.02.013
[21]   尹佳, 詹冬玲, 崔敬爱 , 等. 抗菌肽Japonicin Ⅱ基因的克隆及其在Pichia pastoris中的表达. 中国酿造, 2009,28(12):23-25.
doi: 10.3969/j.issn.0254-5071.2009.12.008
[21]   Yin J, Zhan D L, Cui J A , et al. Cloning and expression of antimicrobial peptide Japonicin Ⅱ in Picha pastoris. China Brewing, 2009,28(12):23-25.
doi: 10.3969/j.issn.0254-5071.2009.12.008
[22]   Vogl T, Glieder A . Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnology, 2013,30(4):385-404.
doi: 10.1016/j.nbt.2012.11.010 pmid: 23165100
[23]   Hsu K H, Pei C, Yeh J Y , et al. Production of bioactive human α-defensin 5 in Pichia pastoris. Journal of General\s&\sapplied Microbiology, 2009,55(5):395-401.
[24]   章华兵 . 融合β防御素基因的构建及其表达. 成都: 四川大学, 2009.
[24]   Zhang H B . Study on the construction and bioactivity of fusion gene of beta defensin. Chengdu: Sichuan University, 2009.
[25]   杨毅 . 新型防御素基因的克隆与原核表达研究. 成都: 四川大学, 2006.
doi: 10.7666/d.y994312
[25]   Yang Y . Construction and prokaryotic expression of novel fusion gene of bovine neutrophil beta defensin 3 and human alpha defensin 3. Chengdu: Sichuan University, 2006.
doi: 10.7666/d.y994312
[26]   程驰 . 新型融合抗菌肽基因的构建及表达活性研究. 成都: 四川大学, 2006.
doi: 10.7666/d.y1002126
[26]   Cheng C . Construction and expression bioactivity of novel fusion gene of antibacterial peptides. Chengdu: Sichuan University, 2006.
doi: 10.7666/d.y1002126
[27]   郑敏 . 融合抗菌肽基因AJI重组真核质粒的构建及其活性研究. 成都: 四川大学, 2008.
[27]   Zheng M . Construction and bioactivity of eukaryotic recombiant plasmid of fusion antibacterial peptide AJI gene. Chengdu: Sichuan University, 2008.
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