中华蜜蜂Apidaecin的重组表达及其抗菌活性
|
摘要
【目的】研究中华蜜蜂(Apis cerana cerana)抗菌肽Apidaecin在枯草芽孢杆菌(Bacillus subtilis)中的重组表达,并验证纯化后的重组抗菌肽Apidaecin在体内和体外是否具有抗菌活性,为开发新型、安全具有抗菌和免疫调节功能的抗菌肽制剂提供理论依据。【方法】以意大利蜜蜂(Apis mellifera ligustica)Apidaecin为模板,克隆出中华蜜蜂抗菌肽Apidaecin,成功构建his-pHT43/Apidaecin表达载体,并参照MoBiTec所提供的制备方法成功制得枯草芽孢杆菌感受态细胞,现配现用,以保证其活性并得以在枯草芽孢杆菌表达系统中重组表达。使用His标签蛋白纯化试剂盒(可溶性蛋白)进行表达蛋白的分离纯化,使用Easy II Protein QuantitativeKit(BCA)试剂盒测定浓度。纯化得到的重组抗菌肽Apidaecin作用于大肠杆菌K88,在体外进行抑菌圈试验和最小浓度抑菌法测定;在体内,以小鼠为试验动物模型,试验组小鼠腹腔注射重组抗菌肽Apidaecin,阴性对照组注射相同体积的生理盐水,阳性对照组注射相同体积的头孢霉素,然后人为感染大肠杆菌K88,感染24 h后对小鼠进行解剖,取得肠道样品和血液样品,并从肠道屏障和免疫功能两个方面探讨重组抗菌肽Apidaecin对感染大肠杆菌K88小鼠的保护作用。使用ELISA试剂盒对染菌小鼠血清免疫球蛋白(IgA、IgG、IgM)与肠道sIgA水平进行测定,采用qRT-PCR法对小鼠肠道紧密连接蛋白基因(claudin-1、ZO-2)以及小鼠肠道细胞因子(促炎因子TNF-α、IFN-γ、IL6和抑炎因子IL10)的转录水平进行测定。【结果】克隆得到的中华蜜蜂Apidaecin含有183bp的碱基,编码60个氨基酸,包含Apidaecin的信号肽序列、一个基础的RR二肽以及抗菌肽Apidaecin的氨基酸序列(内含高度保守的8个氨基酸序列),编码的蛋白质命名为AccApidaecin,其分子量为15.6 kD,等电点为5.33。在IPTG终浓度为3 mmol·L~(-1),诱导温度为30℃,诱导表达12 h后,可从1 L表达上清中纯化得到约20 mg抗菌肽。药敏试验显示,重组抗菌肽Apidaecin在体外与阴性对照相比有明显抑菌圈出现,且测得的最小抑菌浓度为10 mg·L~(-1);在小鼠体内,腹腔注射重组抗菌肽Apidaecin的染菌试验组与注射生理盐水的染菌试验组相比免疫球蛋白含量差异显著(P<0.05),说明腹腔注射重组抗菌肽Apidaecin能够有效缓解由大肠杆菌K88引起的小鼠免疫球蛋白含量的增加;同时,小鼠肠道相关蛋白的基因表达量也差异显著(P<0.05),说明重组抗菌肽Apidaecin能够有效保护被大肠杆菌K88侵染的小鼠肠道。【结论】在枯草芽孢杆菌中能够成功重组表达中华蜜蜂抗菌肽Apidaecin,并且纯化后的中华蜜蜂抗菌肽Apidaecin在体外对大肠杆菌K88有良好的抗菌效果;经腹腔注射进入小鼠体内,能够提高小鼠的免疫机能,有效抵抗大肠杆菌K88对小鼠的侵染。
【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.
【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.
引文
[1]PARACHIN N S,MULDER K C,VIANA A A,DIAS S C,FRANCOO L.Expression systems for heterologous production of antimicrobial peptides.Peptides,2012,38(2):446-456.
[2]S?RENSEN H P,MORTENSEN K K.Advanced genetic strategies for recombinant protein expression in Escherichia coli.Journal of Biotechnology,2005,115(2):113-128.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.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)
[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.
[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.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)
[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.
[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.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)
[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.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)
[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.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)
[30]HARANDI A M,HOLMGREN J.CpG oligodeoxynucleotides and mobilization of innate mucosal immunity:tasks and tactics.Vaccine,2006,24(Suppl.2):S48-S49.
[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.
[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.
[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.
[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.
[2]S?RENSEN H P,MORTENSEN K K.Advanced genetic strategies for recombinant protein expression in Escherichia coli.Journal of Biotechnology,2005,115(2):113-128.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[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.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)
[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.
[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.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)
[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.
[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.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)
[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.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)
[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.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)
[30]HARANDI A M,HOLMGREN J.CpG oligodeoxynucleotides and mobilization of innate mucosal immunity:tasks and tactics.Vaccine,2006,24(Suppl.2):S48-S49.
[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.
[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.
[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.
[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.