基于流式细胞术分选绵羊分泌型IgA包裹的消化道细菌

doi:10.11843/j.issn.0366-6964.2023.07.023

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (7): 2924-2931.doi: 10.11843/j.issn.0366-6964.2023.07.023

基于流式细胞术分选绵羊分泌型IgA包裹的消化道细菌

米慧¹, 彭灿^1*, 贺志雄^1,2*, 谭支良^1,2

1. 中国科学院亚热带农业生态研究所亚热带农业生态过程重点实验室畜禽养殖污染控制与资源化技术国家工程实验室湖南省畜禽健康养殖工程技术研究中心农业部中南动物营养与饲料科学观测实验站动物营养生理与代谢过程湖南省重点实验室中国科学院亚热带农业生态研究所公共技术中心, 长沙 410125;
2. 中国科学院大学, 北京 100049

收稿日期:2022-09-13 出版日期:2023-07-23 发布日期:2023-07-21
通讯作者: 彭灿,主要从事细胞生物学研究;E-mail:pengcan@isa.ac.cn;贺志雄,主要从事幼龄反刍动物营养与免疫研究,E-mail:zxhe@isa.ac.cn
作者简介:米慧(1997-),女,湖南邵阳人,硕士生,主要从事反刍动物营养研究,E-mail:18374962401@163.com
基金资助:
国家自然科学基金（32072760）；湖南创新省份建设专项（2019RS3021）

Separation of Sheep Secretory Immunoglobulin A Coated Bacteria by Flow Cytometry

MI Hui¹, PENG Can^1*, HE Zhixiong^1,2*, TAN Zhiliang^1,2

1. Institutional Center for Shared Technologies and Facilities of Institute of Subtropical Agriculture, Hunan Provincial Key Laboratory of Nutritional Physiology and Metabolic Process, South Central Experimental Station of Animal Nutrition and Feed Science in the Ministry of Agriculture, Hunan Research Center of Livestock & Poultry Sciences, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Key Laboratory for Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China;
2. University of Chinese Academy of Sciences, Beijing 100049, China

Received:2022-09-13 Online:2023-07-23 Published:2023-07-21

摘要/Abstract

摘要： 本研究旨在建立1种基于流式细胞术（flow cytometry，FCM）的反刍动物消化道内容物中分泌型免疫球蛋白A （secretory immunoglobulin A，sIgA）包裹细菌的分选方法。随机选用4只健康、体况良好的成年肉用绵羊，采集回肠内容物制备菌液。对菌液上样浓度、细菌染料4’，6-二脒基-2-苯基吲哚（4’，6-diamidino-2-phenylindole，DAPI）最佳工作浓度和抗IgA抗体最佳使用浓度等进行优化。结果表明，内容物菌液最佳浓度为2.5~5 mg·mL^-1，DAPI最佳工作浓度为5 μg·mL^-1，抗IgA抗体最佳使用量为5 μg。重复性试验表明，该方法相对偏差小于5%，具有良好的稳定性和可重复性。本试验建立的sIgA包裹细菌分选方法选择性强、样品用量少、操作简单，可为反刍动物消化道sIgA包裹细菌的组成及功能等相关研究提供技术支撑。

关键词: 分泌型免疫球蛋白A, 分泌型免疫球蛋白A包裹细菌, 微生物, 流式细胞仪

Abstract: This study aimed to establish a flow cytometry (FCM) method for sorting secretory immunoglobulin A (sIgA) coated bacteria from the digestive tract contents of ruminants. Four healthy adult meat sheep in good physical condition were randomly selected and their ileal contents were collected to prepare the bacterial solution. The loading concentration of the bacterial solution, the working concentration of the bacterial dye 4', 6-diamidino-2-phenylindole (DAPI) and the anti-IgA antibody were optimized. The results showed that the optimal loading concentration of the bacterial solution of the contents was from 2.5 to 5 mg·mL^-1, the best staining concentration of DAPI was 5 μg·mL^-1, and the optimum amount of anti-IgA antibody was 5 μg. The reproducibility test results showed that the relative bias of the proposed method is less than 5% and that it is stable and reproducible. The sorting method of sIgA-coated bacteria established in this study is high selectivity, less sample consumption and easy operation and can provide technical support for related studies of the composition and function of sIgA-coated bacteria in ruminant digestive tracts.

Key words: secretory immunoglobulin A, secretory immunoglobulin A coated bacteria, microbe, flow cytometer

中图分类号:

S826.5

米慧, 彭灿, 贺志雄, 谭支良. 基于流式细胞术分选绵羊分泌型IgA包裹的消化道细菌[J]. 畜牧兽医学报, 2023, 54(7): 2924-2931.

MI Hui, PENG Can, HE Zhixiong, TAN Zhiliang. Separation of Sheep Secretory Immunoglobulin A Coated Bacteria by Flow Cytometry[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2924-2931.

参考文献 34

[1]	CHOLEWIŃSKA P, CZYŻ K, NOWAKOWSKI P, et al.The microbiome of the digestive system of ruminants-a review[J].Anim Health Res Rev, 2020, 21(1):3-14.
[2]	MALMUTHUGE N, GUAN L L.Gut microbiome and omics:A new definition to ruminant production and health[J].Anim Front, 2016, 6(2):8-12.
[3]	SEIKRIT C, PABST O.The immune landscape of IgA induction in the gut[J].Semin Immunopathol, 2021, 43(5):627-637.
[4]	STERLIN D, FADLALLAH J, ADAMS O, et al.Human IgA binds a diverse array of commensal bacteria[J].J Exp Med, 2020, 217(3):e20181635.
[5]	NAKAJIMA A, VOGELZANG A, MARUYA M, et al.IgA regulates the composition and metabolic function of gut microbiota by promoting symbiosis between bacteria[J].J Exp Med, 2018, 215(8):2019-2034.
[6]	MATHIAS A, CORTHÉSY B.Recognition of gram-positive intestinal bacteria by hybridoma-and colostrum-derived secretory immunoglobulin a is mediated by carbohydrates[J].J Biol Chem, 2011, 286(19):17239-17247.
[7]	BUNKER J J, DREES C, WATSON A R, et al.B cell superantigens in the human intestinal microbiota[J].Sci Transl Med, 2019, 11(507):eaau9356.
[8]	PALM N W, DE ZOETE M R, CULLEN T W, et al.Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease[J].Cell, 2014, 158(5):1000-1010.
[9]	BUNKER J J, FLYNN T M, KOVAL J C, et al.Innate and adaptive humoral responses coat distinct commensal bacteria with immunoglobulin A[J].Immunity, 2015, 43(3):541-553.
[10]	KAU A L, PLANER J D, LIU J, et al.Functional characterization of IgA-targeted bacterial taxa from undernourished Malawian children that produce diet-dependent enteropathy[J].Sci Transl Med, 2015, 7(276):276ra24.
[11]	YANG Y, PALM N W.Immunoglobulin A and the microbiome[J].Curr Opin Microbiol, 2020, 56:89-96.
[12]	MCLOUGHLIN K, SCHLUTER J, RAKOFF-NAHOUM S, et al.Host selection of microbiota via differential adhesion[J].Cell Host Microbe, 2016, 19(4):550-559.
[13]	SLACK E, BALMER M L, FRITZ J H, et al.Functional flexibility of intestinal IgA-broadening the fine line[J].Front Immunol, 2012, 3:100.
[14]	OKAI S, USUI F, YOKOTA S, et al.High-affinity monoclonal IgA regulates gut microbiota and prevents colitis in mice[J].Nat Microbiol, 2016, 1(9):16103.
[15]	CULLENDER T C, CHASSAING B, JANZON A, et al.Innate and adaptive immunity interact to quench microbiome flagellar motility in the gut[J].Cell Host Microbe, 2013, 14(5):571-581.
[16]	FADLALLAH J, EL KAFSI H, STERLIN D, et al.Microbial ecology perturbation in human IgA deficiency[J].Sci Transl Med, 2018, 10(439):eaan1217.
[17]	CATANZARO J R, STRAUSS J D, BIELECKA A, et al.IgA-deficient humans exhibit gut microbiota dysbiosis despite secretion of compensatory IgM[J].Sci Rep, 2019, 9(1):13574.
[18]	KOCH C, HARMS H, MVLLER S.Dynamics in the microbial cytome-single cell analytics in natural systems[J].Curr Opin Biotechnol, 2014, 27:134-141.
[19]	CUI M J, QI C, YANG L P, et al.A pregnancy complication-dependent change in sIgA-targeted microbiota during third trimester[J].Food Funct, 2020, 11(2):1513-1524.
[20]	WANG H, LI Y B, WANG A, et al.Rapid, sensitive, and simultaneous detection of three foodborne pathogens using magnetic nanobead-based immunoseparation and quantum dot-based multiplex immunoassay[J].J Food Prot, 2011, 74(12):2039-2047.
[21]	ZHU P X, SHELTON D R, LI S H, et al.Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay[J].Biosens Bioelectron, 2011, 30(1):337-341.
[22]	杜美红, 代凤英, 许迪莘, 等.免疫磁球性能影响因素[J].食品与生物技术学报, 2015, 34(5):501-506.DU M H, DAI F Y, XU D X, et al.Factors effected on performance of immunomagnetic beads for bacteria enrichment[J].Journal of Food Science and Biotechnology, 2015, 34(5):501-506.(in Chinese)
[23]	COMAS-RIU J, RIUS N.Flow cytometry applications in the food industry[J].J Ind Microbiol Biotechnol, 2009, 36(8):999-1011.
[24]	AMBRIZ-AVIÑA V, CONTRERAS-GARDUÑO J A, PEDRAZA-REYES M.Applications of flow cytometry to characterize bacterial physiological responses[J].Biomed Res Int, 2014, 2014:461941.
[25]	RASKOVA KAFKOVA L, BROKESOVA D, KRUPKA M, et al.Secretory IgA N-glycans contribute to the protection against E. coli O55 infection of germ-free piglets[J].Mucosal Immunol, 2021, 14(2):511-522.
[26]	ZHANG H W, QI C, ZHAO Y N, et al.Depletion of gut secretory immunoglobulin A coated Lactobacillus reuteri is associated with gestational diabetes mellitus-related intestinal mucosal barrier damage[J].Food Funct, 2021, 12(21):10783-10794.
[27]	MUHOMAH T A, NISHINO N, KATSUMATA E, et al.High-fat diet reduces the level of secretory immunoglobulin A coating of commensal gut microbiota[J].Biosci Microbiota Food Health, 2019, 38(2):55-64.
[28]	WANG X F, HOWE S, DENG F L, et al.Current applications of absolute bacterial quantification in microbiome studies and decision-making regarding different biological questions[J].Microorganisms, 2021, 9(9):1797.
[29]	刘倩, 刘小杰.流式细胞术检测水中细菌总数的方法研究[J].工业水处理, 2022, 42(5):163-168.LIU Q, LIU X J.Detecting total bacteria number in environmental water by flow cytometry[J].Industrial Water Treatment, 2022, 42(5):163-168.(in Chinese)
[30]	梅仕良.豆制品中菌落总数的流式细胞术检测研究[D].上海:上海师范大学, 2019.MEI S L.The study of bacterial colonies in soybean products by flow cytometry[D].Shanghai:Shanghai Normal University.(in Chinese)
[31]	ANVARIAN A H P, SMITH M P, OVERTON T W.The effects of orange juice clarification on the physiology of Escherichia coli; growth-based and flow cytometric analysis[J].Int J Food Microbiol, 2016, 219:38-43.
[32]	TERASHIMA M, KAMAGATA Y, KATO S.Rapid enrichment and isolation of polyphosphate-accumulating organisms through 4'6-diamidino-2-phenylindole (DAPI) staining with fluorescence-activated cell sorting (FACS)[J].Front Microbiol, 2020, 11:793.
[33]	PIRIYAKARNSAKUL S, TAKARADA K, HEAB K E, et al.Optimal fluorescent-dye staining time for the real-time detection of microbes:A study of Saccharomyces cerevisiae[J].J Appl Microbiol, 2020, 128(6):1694-1702.
[34]	HERNLEM B J, RAVVA S V, SARREAL C Z.Rapid detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous Tetrahymena by flow cytometry[J].Front Cell Infect Microbiol, 2014, 4:57.

基于流式细胞术分选绵羊分泌型IgA包裹的消化道细菌

Separation of Sheep Secretory Immunoglobulin A Coated Bacteria by Flow Cytometry

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