中华蜜蜂Apidaecin的重组表达及其抗菌活性

doi:10.3864/j.issn.0578-1752.2019.04.016

Abstract

Abstract:

【Objective】In order to study the recombinant expression of antimicrobial peptide Apidaecin of Apis cerana cerana in Bacillus subtilis, verify whether the purified recombinant antimicrobial peptide Apidaecin has antibacterial activity in vivo and in vitro or not, and to provide a theoretical basis for the development of new antimicrobial peptide preparations with safe antibacterial and immunomodulatory functions.【Method】The experiment used Apidaecin in Apis mellifera ligustica as templates to clone the antimicrobial peptide Apidaecin in A. c. cerana, and his-pHT43/Apidaecin expression vector was successfully constructed. In addition, the competent cells were successfully prepared according to the method provided by MoBiTec. The cells were prepared and used on demand to ensure the activity. Protein was isolated and purified using 6×His-Tagged Protein Purification Kit (soluble protein), and the Easy II Protein Quantitative Kit (BCA) was used for concentration determination. The purified recombinant antimicrobial peptide Apidaecin acted on Escherichia coli K88. The disk diffusion test and minimum inhibitory concentration (MIC) were conducted in vitro. In vivo, mice were used as experimental animal models, mice in the experimental group were injected with recombinant antibacterial peptide Apidaecin by intraperitoneal injection, mice in the negative control group were injected with normal saline of the same volume, and the positive control group was injected with the same volume of cefotaxime, then artificially infected with E. coli K88. After 24 hours of infection, the mice were dissected, intestinal samples and blood samples were obtained, and the protective effect of Apidaecin on the mice infected with E. coli K88 was discussed from the perspective of intestinal barrier and immune function. Serum immunoglobulin (IgA, IgG, IgM) and intestinal sIgA levels were determined by using ELISA Kit in the experiment. In addition, the expression levels of intestinal tightly-connected protein genes (claudin-1, ZO-2) and intestinal cytokines (proinflammatory cytokines TNF-α, IFN-γ, IL6 and anti-inflammatory factor IL10) were determined by qRT-PCR. 【Result】The Apidaecin in A. c. cerana was cloned, containing 183 bp, encoding 60 amino acids, including signal peptide sequence of Apidaecin, a basic RR dipeptide and amino acid sequence of Apidaecin (containing 8 highly conserved amino acid sequences). The protein encoded is named AccApidaecin, its molecular weight is 15.6 kD and isoelectric point is 5.33. About 20 mg antimicrobial peptides could be purified from 1 L supernatant at the concentration of IPTG was 3 mmol·L ^-1, induction temperature was 30℃, and inducing time was 12 h. The recombinant antibacterial peptide Apidaecin showed obvious antibacterial ring in vitro compared with the negative control, and the measured MIC was 10 mg·L ^-1. In mice, there were significant differences in immunoglobulin between the bacterial test group injected with recombinant antimicrobial peptide Apidaecin by intraperitoneal injection and the bacterial test group injected with saline (P<0.05), which indicated that recombinant Apidaecin could effectively alleviate the increase of immunoglobulin content in mice caused by E.coli K88. The gene expression level of intestinal related proteins in mice was also significantly different (P<0.05), which indicated that the recombinant Apidaecin could effectively protect the intestinal tract of mice infected with E. coli K88.【Conclusion】The antimicrobial peptide Apidaecin in A. c. cerana was successfully expressed in B. subtilis. The purified antibacterial peptide Apidaecin in A. c. cerana has a good antibacterial effect on E. coli K88 in vitro. In addition, it can be injected into mice through abdominal cavity to improve the immune function of mice and effectively resist the infection of E. coli K88 to mice.

Key words: Apis cerana cerana, antimicrobial peptide, recombinant expression, Escherichia coli, antimicrobial activity

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.

Figures/Tables 8

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

References 34

[1]	PARACHIN N S, MULDER K C, VIANA A A, DIAS S C, FRANCO O L . Expression systems for heterologous production of antimicrobial peptides. Peptides, 2012,38(2):446-456. doi: 10.1016/j.peptides.2012.09.020
[2]	SØRENSEN H P, MORTENSEN K K . Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology, 2005,115(2):113-128. doi: 10.1016/j.jbiotec.2004.08.004 pmid: 15607230
[3]	DIGAN M E, TSCHOPP J, GRINNA L, LAIR S V, CRAIG W S, VELICELEBI G, SIEGEL R, DAVIS G R, THILL G P . Secretion of heterologous proteins from the methylotrophic yeast, Pichia pastoris. Developments in Industrial Microbiology, 1988,29:59-65.
[4]	LI W, ZHOU X, LU P . Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Research in Microbiology, 2004,155(8):605-610. doi: 10.1016/j.resmic.2004.05.002 pmid: 15380546
[5]	VAN DIJL J M, HECKER M . Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microbial Cell Factories, 2013,12:3.
[6]	WESTERS L, WESTERS H, QUAX W J . Bacillus subtilis, as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochimica et Biophysica Acta Molecular Cell Research, 2004,1694(1/3):299-310. doi: 10.1016/j.bbamcr.2004.02.011 pmid: 15546673
[7]	BIEN T L T, TSUJI S, TANAKA K, TAKENAKA S, YOSHIDA K I . Secretion of heterologous thermostable cellulases in Bacillus subtilis. The Journal of General and Applied Microbiology, 2014,60(5):175-182.
[8]	华婷 . 蜜蜂抗菌肽Apidaecins在不同表达系统中融合表达的研究[D]. 北京: 中国农业科学院, 2017.
	HUA T . Study on the fusion expression of antibacterial peptide Apidaecins in various expression systems[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017. (in Chinese)
[9]	CASTEELS P, AMPE C, JACOBS F, VAECK M, TEMPST P . Apidaecins: antibacterial peptides from honeybees. The EMBO Journal, 1989,8(8):2387-2391. doi: 10.1002/j.1460-2075.1989.tb08368.x pmid: 2676519
[10]	CASTEELS P, AMPE C, RIVIERE L, VAN DAMME J, ELICONE C, FLEMING M, JACOBS F, TEMPST P . Isolation and characterization of abaecin, a major antibacterial response peptide in the honeybee (Apis mellifera). European Journal of Biochemistry, 1990,187(2):381-386. doi: 10.1111/j.1432-1033.1990.tb15315.x pmid: 2298215
[11]	CASTEELS P, AMPE C, JACOBS F, TEMPST P . Functional and chemical characterization of hymenoptaecin, an antibacterial polypeptide that is infection-inducible in the honeybee (Apis mellifera). The Journal of Biological Chemistry, 1993,268(10):7044-7054.
[12]	CASTEELS P, TEMPST P . Apidaecin-type peptide antibiotics function through a non-poreforming mechanism involving stereospecificity. Biochemical and Biophysical Research Communications, 1994,199(1):339-345. doi: 10.1006/bbrc.1994.1234 pmid: 8123032
[13]	WANG K, QI Y, YI S, PEI Z, PAN N, HU G . Mouse duodenum as a model of inflammation induced by enterotoxigenic Escherichia coli K88. Journal of Veterinary Research, 2016,60(1):19-23.
[14]	CASTEELS-JOSSON K, CAPACI T, CASTEELS P, TEMPST P . Apidaecin multipeptide precursor structure: a putative mechanism for amplification of the insect antibacterial response. The EMBO Journal, 1993,12(4):1569-1578. doi: 10.1002/j.1460-2075.1993.tb05801.x pmid: 8467807
[15]	AITKEN R, GILCHRIST J, SINCLAIR M C . Vectors to facilitate the creation of translational fusions to the maltose-binding protein of Escherichia coli. Gene, 1994,144(1):69-73. doi: 10.1016/0378-1119(94)90205-4 pmid: 8026760
[16]	MURTHY T V S . Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris, facilitates easy purification. Biotechnology Letters, 2004,26(5):443-449. doi: 10.1128/AAC.46.5.1503-1509.2002 pmid: 15104145
[17]	PENG C C, CHEN J C, SHYU D J, CHEN M J, TZEN J T . A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides. Journal of Biotechnology, 2004,111(1):51-57. doi: 10.1016/j.jbiotec.2004.03.013 pmid: 15196769
[18]	XU X, JIN F, YU X, REN S, HU J, ZHANG W . High-level expression of the recombinant hybrid peptide cecropinA (1-8)-magainin2 (1-12) with an ubiquitin fusion partner in Escherichia coli. Protein Expression and Purification, 2007,55(1):175-182. doi: 10.1016/j.pep.2007.04.018 pmid: 17572103
[19]	BANG S K, KANG C S, HAN M D, BANG I S . Expression of recombinant hybrid peptide hinnavin II/ α-melanocyte-stimulating hormone in Escherichia coli: Purification and characterization. Journal of Microbiology, 2010,48(1):24-29.
[20]	FENG X, LIU C, GUO J, SONG X, LI J, XU W, LI Z . Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33. Applied Microbiology and Biotechnology, 2012,95(5):1191-1198. doi: 10.1007/s00253-011-3816-z pmid: 22189867
[21]	施文, 马瑞, 周建业, 陈莉娅, 马媛媛, 黄慧敏, 张小凤, 易根云, 李志强 . Apidaecin型抗菌肽在毕赤酵母菌中的基因工程表达. 口腔医学研究, 2017,33(5):471-474. doi: 10.13701/j.cnki.kqyxyj.2017.05.002
	SHI W, MA R, ZHOU J Y, CHEN L Y, MA Y Y, HUANG H M, ZHANG X F, YI G Y, LI Z Q . Expression and identification of Apidaecin in Pichia pastoris. Journal of Oral Science Research, 2017,33(5):471-474. (in Chinese) doi: 10.13701/j.cnki.kqyxyj.2017.05.002
[22]	黄玉明 . 中华蜜蜂apidaecin基因在乳酸乳球菌中的融合表达[D]. 广州: 中山大学, 2006.
	HUANG Y M . Fusion expression of Apis cerana cerana apidaecin gene in Lactococcus lactis[D]. Guangzhou: Sun Yat-Sen University, 2006. (in Chinese)
[23]	LI W F, MA G X, ZHOU X X . Apidaecin-type peptides: biodiversity, structure-function relationships and mode of action. Peptides, 2006,27(9):2350-2359. doi: 10.1016/j.peptides.2006.03.016 pmid: 16675061
[24]	马军宏, 于向阳, 张楠, 周振理 . 紧密连接蛋白与肠黏膜屏障损伤研究进展. 中国中西医结合外科杂志, 2015,21(1):104-105. doi: 10.3969/j.issn.1007-6948.2015.01.037
	MA J H, YU X Y, ZHANG N, ZHOU Z L . The progress of closely connected protein and intestinal mucosal barrier damage. Chinese Journal of Surgery of Integrated Traditional and Western Medicine, 2015,21(1):104-105. (in Chinese) doi: 10.3969/j.issn.1007-6948.2015.01.037
[25]	TORRES M I, RÍOS A . Current view of the immunopathogenesis in inflammatory bowel disease and its implications for therapy. World Journal of Gastroenterology, 2008,14(13):1972-1980. doi: 10.3748/wjg.14.1972 pmid: 18395894
[26]	齐珂珂, 吴杰, 徐子伟 . 聚乙二醇修饰猪胰高血糖素样肽-2对试验性结肠炎小鼠肠道紧密连接蛋白和炎性因子基因表达的影响. 动物营养学报, 2014,26(9):2745-2751. doi: 10.3969/j.issn.1006-267x.2014.09.037
	QI K K, WU J, XU Z W . Effects of polyethylene glycosylation porcine glucagon-like peptide-2 on gene expression of tight junction proteins and inflammatory cytokines in a murine model of experimental colitis. Chinese Journal of Animal Nutrition, 2014,26(9):2745-2751. (in Chinese) doi: 10.3969/j.issn.1006-267x.2014.09.037
[27]	余树培 . K88ac⁺大肠杆菌减毒株的构建及其在小鼠体内的初步应用 [D]. 扬州: 扬州大学, 2016.
	YU S P . Construction of K88ac⁺ ETEC attenuated strain and its preliminary application in vivo of mouse [D]. Yangzhou: Yangzhou University, 2016. (in Chinese)
[28]	罗献梅, 陈代文, 张克英 . 乳铁蛋白及其活性肽的营养生理作用及应用前景. 饲料工业, 2005,26(2):5-9. doi: 10.3969/j.issn.1001-991X.2005.02.002
	LUO X M, CHEN D W, ZHANG K Y . Nutritional functions and application prospect of lactoferrin and active polypeptide. Feed Industry, 2005,26(2):5-9. (in Chinese) doi: 10.3969/j.issn.1001-991X.2005.02.002
[29]	单体中, 汪以真 . 重组猪乳铁蛋白(rPLF)对断奶仔猪血清IL-1、IL-2水平的影响. 中国兽药杂志, 2005,39(10):6-8. doi: 10.3969/j.issn.1002-1280.2005.10.002
	SHAN T Z, WANG Y Z . Effects of the recombinant porcine lactoferrin on the interleukin-1 and interleukin-2 in serum of weanling pigs. Chinese Journal of Veterinary Drug, 2005,39(10):6-8. (in Chinese) doi: 10.3969/j.issn.1002-1280.2005.10.002
[30]	HARANDI A M, HOLMGREN J . CpG oligodeoxynucleotides and mobilization of innate mucosal immunity: tasks and tactics. Vaccine, 2006,24(Suppl. 2):S48-S49. doi: 10.1016/j.vaccine.2005.01.118 pmid: 16823923
[31]	CHOI H, RANGARAJAN N, WEISSHAAR J C . Lights, camera, action! Antimicrobial peptide mechanisms imaged in space and time. Trends in Microbiology, 2016,24(2):111-122. doi: 10.1016/j.tim.2015.11.004 pmid: 4733415
[32]	NGUYEN L T, HANEY E F, VOGEL H J . The expanding scope of antimicrobial peptide structures and their modes of action. Trends in Biotechnology, 2011,29(9):464-472. doi: 10.1016/j.tibtech.2011.05.001 pmid: 21680034
[33]	DAGAN A, EFRON L, GAIDUKOV L, MOR A, GINSBURG H . In vitro antiplasmodium effects of dermaseptin S4 derivatives. Antimicrobial Agents and Chemotherapy, 2002,46(4):1059-1066. doi: 10.1016/j.physbeh.2009.07.008 pmid: 127115
[34]	YONEYAMA F, IMURA Y, OHNO K, ZENDO T, NAKAYAMA J, MATSUZAKI K, SONOMOTO K . Peptide-lipid huge toroidal pore, a new antimicrobial mechanism mediated by a lactococcal bacteriocin, lacticin Q. Antimicrobial Agents and Chemotherapy, 2009,53(8):3211-3217. doi: 10.1128/AAC.00209-09 pmid: 19470516

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

基因名称 Gene name	引物序列 Primer sequence (5′-3′)	引物用途 Description	登录号 GenBank accession number
API-F	TGTGGGTTGAATAACTATTGATAA	cDNA sequence primer, forward	NM_001011613.1
API-R	GTTATTTCACGTGCTTCATATTC	cDNA sequence primer, reverse	NM_001011613.1
GAPDH-F	GGTTGTCTCCTGCGACTTCA	Standard control primer, forward	NM_001289726.1
GAPDH-R	TGGTCCAGGGTTTCTTACTCC	Standard control primer, reverse	NM_001289726.1
IL10-F	ACTGCTATGCTGCCTGCTCT	Real-time PCR primer, forward	NM_010548.2
IL10-R	GACTGGGAAGTGGGTGCAGT	Real-time PCR primer, reverse	NM_010548.2
TNF-α-F	CCAGCCGATGGGTTGTACCT	Real-time PCR primer, forward	NM_001278601.1
TNF-α-R	CAAATCGGCTGACGGTGTGG	Real-time PCR primer, reverse	NM_001278601.1
IL6-F	TGGGACTGATGCTGGTGACA	Real-time PCR primer, forward	NM_031168.2
IL6-R	ACAGGTCTGTTGGGAGTGGT	Real-time PCR primer, reverse	NM_031168.2
ZO-2-F	CCACCTCGCACGCATCACAG	Real-time PCR primer, forward	XM_017318137.1
ZO-2-R	TGGTCCTTCACCTCTGAGCACTAC	Real-time PCR primer, reverse	XM_017318137.1
Claudin-1-F	TCGGCTCCATCGTCAGCACTG	Real-time PCR primer, forward	NM_016674.4
Claudin-1-R	AGATGGCCTGAGCGGTCACG	Real-time PCR primer, reverse	NM_016674.4
IFN-γ-F	GGCTCTGGAGGCTGGAGGAAG	Real-time PCR primer, forward	NM_008337.4
IFN-γ-R	TGATAGGCGGTGAGGCTACAAGG	Real-time PCR primer, reverse	NM_008337.4

分组 Group	步骤1 Step 1 （第1—6天腹腔注射 Intraperitoneal injection at 1st-6th days）	步骤2 Step 2 （第6天腹腔注射后1 h再注射 Repeated intraperitoneal injection was performed 1 h after the first injection at 6th day）
A：正常对照组 Normal control group	0.5 mL生理盐水0.5 mL Saline	0.5 mL生理盐水0.5 mL Saline
B：pHT43空载体 pHT43 Empty carrier	0.5 mL pHT43	0.5 mL生理盐水 0.5 mL Saline
C：染菌对照组Infection control group	0.5 mL生理盐水0.5 mL Saline	0.5 mL菌悬液0.5 mL Bacteria suspension
D：pHT43空载体染菌组 pHT43 Empty carrier infection group	0.5 mL pHT43	0.5 mL菌悬液0.5 mL Bacteria suspension
E：20 mg·L^-1重组Apidaecin染菌组 20 mg·L^-1 Recombinant Apidaecin infection group	0.5 mL重组Apidaecin 0.5 mL Recombinant Apidaecin	0.5mL菌悬液0.5 mL Bacteria suspension
F：150 mg·L^-1头孢噻肟染菌组 150 mg·L^-1 Cefotaxime infection group	0.5 mL头孢噻肟溶液 0.5 mL Cefotaxime solution	0.5 mL菌悬液0.5 mL Bacteria suspension

Recombinant Expression and Antimicrobial Activity of Apidaecin in Apis cerana cerana

RichHTML

PDF

Abstract

Cite this article

share this article

Figures/Tables 8

References 34

Related Articles 15

Metrics

Comments

Recommended 0

[1]	CAO Peng, ZHOU JinHuan, WANG XinLiang, LI ChuXin, LI JiaXIN, JIANG Pei, LIU JinXiang, SONG Zhen. Optimization and Application of Rapid Evaluation System for Citrus Huanglongbing Resistance Mediated by Agrobacterium rhizogenes [J]. Scientia Agricultura Sinica, 2024, 57(16): 3182-3191.
[2]	QIU YiLei,WU Fan,ZHANG Li,LI HongLiang. Effects of Sublethal Doses of Imidacloprid on the Expression of Neurometabolic Genes in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(8): 1685-1694.
[3]	LIU Jiao,LIU Chang,CHEN Jin,WANG MianZhi,XIONG WenGuang,ZENG ZhenLing. Distribution Characteristics of Prophage in Multidrug Resistant Escherichia coli as well as Its Induction and Isolation [J]. Scientia Agricultura Sinica, 2022, 55(7): 1469-1478.
[4]	ZHAO HuiTing,PENG Zhu,JIANG YuSuo,ZHAO ShuGuo,HUANG Li,DU YaLi,GUO LiNa. Expression and Binding Properties of Odorant Binding Protein AcerOBP7 in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(3): 613-624.
[5]	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.
[6]	ZHANG Li,ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang. Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(12): 2461-2471.
[7]	ZHANG AiJing,LI LinQiong,WANG PengJie,GAO YuLong. Effects of Heat Stress on Cell Membrane and Membrane Protein of Escherichia coli [J]. Scientia Agricultura Sinica, 2020, 53(5): 1046-1057.
[8]	DU JiGe, XUE Qi, ZHU Zhen, LI QiHong, YIN ChunSheng, YAO WenSheng, KANG Kai, CHEN XiaoYun. Expression and Evaluation of Protective Efficacy of No-toxic Clostridium perfringens ε Toxin Derivative [J]. Scientia Agricultura Sinica, 2018, 51(11): 2206-2215.
[9]	WEI Shao-peng, GUO Zheng, JI Zhi-qin. Identification and Antimicrobial Ingredients of an Endophytic Actinomycete Strain H21 from Berberis thunbergii [J]. Scientia Agricultura Sinica, 2015, 48(6): 1095-1102.
[10]	ZHOU Cai-Yuan, ZHANG Ming-Yue, HAN Zong-Xi, SHAO Yu-Hao, LIU Sheng-Wang, MA De-Ying. Isolation, Characterization, and Expression of Goose Avian β-Defensin 10 [J]. Scientia Agricultura Sinica, 2012, 45(5): 999-1009.
[11]	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.
[12]	. Integrated Expression of mleA Gene in Saccharomyces cerevisiae [J]. Scientia Agricultura Sinica, 2009, 42(4): 1372-1377 .
[13]	. mRNA Cloning, Tissue Distribution and Expression of Duck Avian Beta-Defensin 9 in E. coli [J]. Scientia Agricultura Sinica, 2009, 42(4): 1406-1412 .
[14]	. 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 .
[15]	. Chitosan Inhibiting the Growth of Phytopathogenic Fungi and Control of Postharvest Diseases of Fruits [J]. Scientia Agricultura Sinica, 2009, 42(2): 626-635 .