Prokaryotic Tandem Expression and Antibody Preparation of Subtilomycin Structural Gene subA from Bacillus subtilis SX3411

doi:10.7668/hbnxb.20192269

Abstract

Abstract: In order to improve the antigenicity of the expression product of Subtilomycin precursor gene of Bacillus subtilis SX3411, a three-times repeated tandem 3×subA gene was synthesized by chemical synthesis method. The prokaryotic expression vector of 3×subA was constructed and transferred into Escherichia coli.The results showed that the 3×subA gene was expressed in E. coli. SDS-PAGE was used to purify the E. coli expressed SubA. After immunizing rabbits, the antibody anti-3×SubA was prepared and had a high titer. The intracellular expression of Subtilomycin precursor polypeptide subA was detected by Western Blotting. The results showed that B. subtilis SX3411 was cultured at 30℃ for different time, and there were more subA expressions in the cells of B. subtilis 3411 at about the 10th and 12th hour. In conclusion, the fusion expression product 3×SubA obtained by tandem expression of the Subtilomycin precursor gene subA has good antigenicity, and the obtained antibody anti-3×SubA can be used for the detection and analysis of Subtilomycin precursors in B subtilis SX3411.

Key words: Bacillus subtilis SX3411, Lantibiotics, Subtilomycin, SubA protein expression, Antibody preparation

CLC Number:

TS201.3

FENG Lei, WEI Weiqun, ZHONG Xueqing, FU Mingjia, XIAO Shiping, YE Dexiao, HUANG Ziyan. Prokaryotic Tandem Expression and Antibody Preparation of Subtilomycin Structural Gene subA from Bacillus subtilis SX3411[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(S1): 347-352. doi: 10.7668/hbnxb.20192269.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20192269

http://www.hbnxb.net/EN/Y2021/V36/IS1/347

References

[1] Drider D,Fimland G,Héchard Y,McMullen L M,Prévost H. The continuing story of class IIa bacteriocins[J]. Microbiology and Molecular Biology Reviews,2006,70(2):564-582.doi:10.1128/mmbr.00016-05.
[2] Todorov S D. Bacteriocins from Lactobacillus plantarum -production,genetic organization and mode of action:Produção,organização genética e modo de ação[J]. Brazilian Journal of Microbiology,2009,40(2):209-221.doi:10.1590/S1517-83822009000200001.
[3] Zgheib H,Drider D,Belguesmia Y. Broadening and enhancing bacteriocins activities by association with bioactive substances[J]. International Journal of Environmental Research and Public Health,2020,17(21):7835.doi:10.3390/ijerph17217835.
[4] Naskar A,Kim K S. Potential novel food-related and biomedical applications of nanomaterials combined with bacteriocins[J]. Pharmaceutics,2021,13(1):86.doi:10.3390/pharmaceutics13010086.
[5] Karczewski J,Krasucki S P,Asare-Okai P N,Diehl C,Friedman A,Brown C M,Maezato Y,Streatfield S J. Isolation,characterization and structure elucidation of a novel lantibiotic from Paenibacillus sp[J]. Frontiers in Microbiology,2020,11:598789.doi:10.3389/fmicb.2020.598789.
[6] Karczewski J,Brown C M,Maezato Y,Krasucki S P,Streatfield S J. Efficacy of a novel lantibiotic,CMB001,against MRSA[J]. Journal of Antimicrobial Chemotherapy,2021,76(6):1532-1538.doi:10.1093/jac/dkab040.
[7] Singh M,Chaudhary S,Sareen D. Roseocin,a novel two-component lantibiotic from an actinomycete[J]. Molecular Microbiology,2020,113(2):326-337.doi:10.1111/mmi.14419.
[8] Vukomanović M,Žunič V,Kunej Š,Jančar B,Jeverica S,Podlipec R,Suvorov D. Nano-engineering the antimicrobial spectrum of lantibiotics:Activity of nisin against gram negative bacteria[J]. Scientific Reports,2017,7:4324.doi:10.1038/s41598-017-04670-0.
[9] Li Q,Montalban-Lopez M,Kuipers O P. Increasing the antimicrobial activity of nisin-based lantibiotics against gram-negative pathogens[J]. Applied and Environmental Microbiology,2018,84(12):e00052-18.doi:10.1128/aem.00052-18.
[10] Zaschke-Kriesche J,Reiners J,Lagedroste M,Smits S H J. Influence of nisin hinge-region variants on lantibiotic immunity and resistance proteins[J]. Bioorganic & Medicinal Chemistry,2019,27(17):3947-3953.doi:10.1016/j.bmc.2019.07.014.
[11] Geng M X,Smith L. Modifying the lantibiotic mutacin 1140 for increased yield,activity,and stability[J]. Applied and Environmental Microbiology,2018,84(15):e00830-18.doi:10.1128/aem.00830-18.
[12] Kers J A,Sharp R E,Defusco A W,Park J H,Xu J,Pulse M E,Weiss W J,Handfield M. Mutacin 1140 lantibiotic variants are efficacious against Clostridium difficile infection[J]. Frontiers in Microbiology,2018,9:415.doi:10.3389/fmicb.2018.00415.
[13] Osipova I G,Sorokulova I B,Tereshkina N V,Grigor'eva L V. Safety of bacteria of the genus Bacillus,forming the base of some probiotics[J]. Zhurnal Mikrobiologii, Epidemiologii Immunobiologii,1998(6):68-70.
[14] 李佳骏,王振华,胡正茂,唐慧琴,谷笑笑,廖成斌,潘康成. 重组枯草芽孢杆菌SE1对肉鸡生长性能、肠道消化酶活性和菌群的影响[J].动物营养学报,2018,30(4):1582-1591.doi:10.3969/j.issn.1006-267x.2018.04.044.
Li J J,Wang Z H,Hu Z M,Tang H Q,Gu X X,Liao C B,Pan K C. Effects of recombinant Bacillus subtilis SE1 on growth performance,intestinal digestive enzyme activities and microflora of broilers[J]. Chinese Journal of Animal Nutrition,2018,30(4):1582-1591.
[15] 于洁,赵敏孟,龚道清. 枯草芽孢杆菌在畜禽生产中的研究进展[J].中国饲料,2020(19):6-11.doi:10.15906/j.cnki.cn11-2975/s.20201902.
Yu J,Zhao M M,Gong D Q. Research progress in application of Bacillus subtilis on animal production[J]. China Feed,2020(19):6-11.
[16] 黄亚丽,郑立伟,黄媛媛,贾振华,宋水山,李再兴. 枯草芽孢杆菌菌剂不同施用方式对甜瓜土壤微生物多样性及生长的影响[J].生物工程学报,2020,36(12):2644-2656.doi:10.13345/j.cjb.200379.
Huang Y L,Zheng L W,Huang Y Y,Jia Z H,Song S S,Li Z X. Effects of different application methods of Bacillus subtilis agent on soil microbial diversity and growth of muskmelon[J]. Chinese Journal of Biotechnology,2020,36(12):2644-2656.
[17] 周亮成,李运,李卓苗,林培炯,钟国华. 枯草芽孢杆菌BSF01菌剂制备及对高效氯氰菊酯的降解效果[J].华南农业大学学报,2018,39(3):54-59.
Zhou L C,Li Y,Li Z M,Lin P J,Zhong G H. Preparation of bacterial agent based on Bacillus subtilis BSF01 and its biodegradation effect on beta-cypermethrin[J]. Journal of South China Agricultural University,2018,39(3):54-59.
[18] Leclère V,Béchet M,Adam A,Guez J S,Wathelet B,Ongena M,Thonart P,Gancel F,Chollet-Imbert M,Jacques P. Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities[J]. Applied and Environmental Microbiology,2005,71(8):4577-4584.doi:10.1128/AEM.71.8.4577-4584.2005.
[19] Stein T. Bacillus subtilis antibiotics:Structures,syntheses and specific functions[J]. Molecular Microbiology,2005,56(4):845-857.doi:10.1111/j.1365-2958.2005.04587.x.
[20] Chu F,Kearns D B,McLoon A,Chai Y R,Kolter R,Losick R. A novel regulatory protein governing biofilm formation in Bacillus subtilis[J]. Molecular Microbiology,2008,68(5):1117-1127.doi:10.1111/j.1365-2958.2008.06201.x.
[21] Heinzmann S,Entian K D,Stein T. Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin[J]. Applied Microbiology and Biotechnology,2006,69(5):532-536.doi:10.1007/s00253-005-0023-9.
[22] Kleerebezem M,Bongers R,Rutten G,de Vos W M,Kuipers O P. Autoregulation of subtilin biosynthesis in Bacillus subtilis:The role of the spa-box in subtilin-responsive promoters[J]. Peptides,2004,25(9):1415-1424.doi:10.1016/j.peptides.2003.11.025.
[23] Chatterjee C,Paul M,Xie L L,van der Donk W A. Biosynthesis and mode of action of lantibiotics[J]. Chemical Reviews,2005,105(2):633-684.doi:10.1021/cr030105v.
[24] Luo Y,Helmann J D. Extracytoplasmic function σ factors with overlapping promoter specificity regulate sublancin production in Bacillus subtilis[J]. Journal of Bacteriology,2009,191(15):4951-4958.doi:10.1128/jb.00549-09.
[25] Zheng G L,Yan L Z,Vederas J C,Zuber P. Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin[J]. Journal of Bacteriology,1999,181(23):7346-7355.doi:10.1128/jb.181.23.7346-7355.1999.
[26] Stein T,Düsterhus S,Stroh A,Entian K D. Subtilosin production by two Bacillus subtilis subspecies and variance of the sbo-alb cluster[J]. Applied and Environmental Microbiology,2004,70(4):2349-2353.doi:10.1128/aem.70.4.2349-2353.2004.
[27] Phelan R W,Barret M,Cotter P D,O'Connor P M,Chen R,Morrissey J P,Dobson A D W,O'Gara F,Barbosa T M. Subtilomycin:a new lantibiotic from Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans[J]. Marine Drugs,2013,11(6):1878-1898.doi:10.3390/md11061878.
[28] Deng Y,Li C Z,Zhu Y G,Wang P X,Qi Q D,Fu J J,Peng D H,Ruan L F,Sun M. ApnI,a transmembrane protein responsible for subtilomycin immunity,unveils a novel model for lantibiotic immunity[J]. Applied and Environmental Microbiology,2014,80(20):6303-6315.doi:10.1128/AEM.02280-14.
[29] Deng Y,Chen H Q,Li C Z,Xu J Y,Qi Q D,Xu Y Y,Zhu Y G,Zheng J S,Peng D H,Ruan L F,Sun M. Endophyte Bacillus subtilis evade plant defense by producing lantibiotic subtilomycin to mask self-produced flagellin[J]. Communications Biology,2019,2:368.doi:10.1038/s42003-019-0614-0.
[30] 李晓然,叶德晓,付鸣佳,钟雪晴,肖世平,杨志海. 枯草芽孢杆菌SX3411产羊毛硫细菌素subtilomycin的初步鉴定与理化特性分析[J].食品与发酵工业,2019,45(12):46-54.doi:10.13995/j.cnki.11-1802/ts.020116.
Li X R,Ye D X,Fu M J,Zhong X Q,Xiao S P,Yang Z H. Identification and physicochemical characterization of lantibiotic subtilomycin produced by Bacillus subtilis SX3411[J]. Food and Fermentation Industries,2019,45(12):46-54.

Please choose a citation manager

Content to export