Preparation and In Vitro Inhibitory Effects of Specific Tripple Anti-vibrio Egg Yolk Antibody
-
摘要:
目的 制备抗弧菌三联特异性卵黄抗体，并研究其对创伤弧菌（Vibrio vulnificus）、河流弧菌（Vibrio fluvialis）和霍乱弧菌（Vibrio cholerae）的体外抑菌作用，为抗生素替代品的研发以及弧菌混合感染的防治提供参考。 方法 制备创伤弧菌、河流弧菌和霍乱弧菌三联灭活疫苗，免疫蛋鸡，制备特异性卵黄抗体（IgY），并检测IgY的效价、纯度和特异性。通过免疫荧光、扫描电镜和体外抑菌试验初步探究制备的三联特异性卵黄抗体对3种弧菌的体外抑菌效果。 结果 使用间接ELISA检测特异性IgY效价，分别以创伤弧菌、霍乱弧菌和河流弧菌作为抗原，特异性IgY效价峰值高达1∶ 51200 ，可持续4～5周。对水稀释-盐析分离纯化的IgY进行SDS-PAGE 分析，可见IgY的重链和轻链，且蛋白杂带逐渐减少，纯度逐渐变高。免疫印迹分析发现，特异性IgY可以和抗原发生特异性结合，出现IgY的蛋白条带。免疫荧光试验结果表明，特异性IgY可以与3种弧菌发生特异性结合，出现明显的菌体凝集。扫描电镜观察发现，特异性IgY处理过的3种弧菌，菌体黏附，菌体细胞结构遭到破坏。体外抑菌试验发现，特异性IgY对3种弧菌的体外生长繁殖具有明显的抑制作用，抑菌效果随抗体用量增加而增强。结论 本研究制备的抗弧菌三联特异性卵黄抗体对创伤弧菌、河流弧菌和霍乱弧菌有较强的抑制作用，在弧菌混合感染的防治和开发新型抗生素替代品方面具有很大的潜力。 Abstract:Objective In vitro inhibitory effects of the prepared specific anti-vibrio antibody on Vibrio vulnificus, V. fluvialis, and V. cholerae were studied for the development of a bioagent to prevent and treat infections caused by the viruses. Method Trivalent inactivated vaccine against the three specific viruses were prepared to immunize hens, and eggs collected for producing the specific IgY. Potency, purity, and specificity of the IgY were determined. Antiviral effects of the antibody were tested by immunofluorescence electron microscopy, scanning electron microscopy, and an in vitro test. Result Indirect ELISA detected the specific IgY with peak titers as high as 1: 51200 that sustained for 4–5 weeks when the target viruses were used as antigens. The SDS-PAGE analysis on the IgY purified by means of water dilution and salt precipitation showed a gradual rise in purity, reduction in protein impurities, and increase in visible heavy and light bands. The immunoblotting of the specific IgY showed its binding to the corresponding antigens with appearance of the protein bands. The immunofluorescence electron microscope displayed apparent agglutination on the bindings to the three viruses. And furthermore, a scanning electron microscopic image of the viruses treated with the specific IgY detailed the adhesion of the antibody to and structural disruption of the virus cells. Finally, significant in vitro, dose-dependent inhibitory effects of the specific IgY on the growth and proliferation of target vibrio strains were observed.Conclusion The prepared specific triple anti-vibrio egg yolk antibody exhibited significant in vitro inhibitory effects on V. vulnificus, V. fluvialis, and V. cholerae. It seemed plausible that the application could become an effective alternative to antibiotic treatment in preventing and controlling the diseases caused by these viruses. -
Key words:
- egg yolk antibody /
- Vibrio vulnificus /
- Vibrio fluvialis /
- Vibrio cholerae /
- inhibition
-
图 1 特异性IgY效价变化趋势
a：创伤弧菌；b：河流弧菌；c：霍乱弧菌。4个红色箭头分别表示初次免疫和3次加强免疫，每次间隔10 d。
Figure 1. Change on titer of specific IgY
a: V. vulnificus; b: V. fluvialis; c: V. cholerae. Four red arrows pointed at one initial immunization and 3 booster immunizations separated by 10 d.
图 2 IgY不同分离纯化阶段的SDS-PAGE
M：预染蛋白Marker；1：水溶性组分；2：第一次盐析；3：第二次盐析。
Figure 2. SDS-PAGE of IgY at different stages of separation and purification
M: pre-stained protein marker; 1: water soluble fractions; 2: the 1st salting out; 3: the 2nd salting out.
图 3 免疫印迹分析
a： 创伤弧菌；b：河流弧菌；c：霍乱弧菌；M：预染蛋白Marker；1：特异性IgY；2：非特异性IgY。
Figure 3. Western blot analysis
a: V. vulnificus; b: V. a fluvialis; c: V. cholerae. M: pre-stained protein marker; 1: specific IgY; 2: non-specific IgY;
图 4 特异性IgY对菌体细胞形态结构的影响
1、3、5：特异性IgY处理组；2、4、6：非特异性IgY处理组。1～2：创伤弧菌；3～4：河流弧菌；5～6：霍乱弧菌。
Figure 4. Effect of specific IgY on morphological structure of virus cells
1, 3, and 5: Specific IgY treatment group; 2, 4, and 6: non-treatment group; 1 and 2: V. vulnificus；3 and 4: V. fluvialis；5 and 6: V. cholerae.
图 5 免疫荧光电镜观察特异性IgY 与致病菌的结合情况
1、4、7：特异性IgY处理组；2、5、8：非特异性IgY处理组；3、6、9：空白对照组。1～3：创伤弧菌；4～6：河流弧菌；7～9：霍乱弧菌。
Figure 5. Binding of specific IgY to pathogenic vibrio observed under immunofluorescence electron microscope
1,4, and 7: specific IgY treatment group; 2, 5, and 8: non-treatment group; 3, 6, and 9: blank control group; 1–3: V. vulnificus；4–6: V. fluvialis；7–9: V. cholerae.
图 6 特异性IgY对致病菌体外生长抑制效果
a：创伤弧菌；b：河流弧菌；c：霍乱弧菌。数据以mean ± SD ( n = 3) 表示；***：表示同一时间不同浓度特异性IgY与不加IgY的空白组之间差异显著（P ＜ 0.001）。
Figure 6. In vitro inhibitory effect of specific IgY on growth of pathogenic vibrio
a: V. vulnificus; b: V. fluvialis; c: V. cholerae. Data are presented as means ± SD (n = 3)； ***: significant difference between different concentrations of specific IgY and blank without IgY at same time (P＜0.001)。
-
[1] 邓益琴. 水产动物弧菌病及其生物防治研究进展 [J]. 大连海洋大学学报, 2023, 38(4):553−563.DENG Y Q. Progress in research on vibriosis and biological control in animals in aquaculture: A review [J]. Journal of Dalian Ocean University, 2023, 38(4): 553−563. (in Chinese) [2] ELGENDY M Y, ABDELSALAM M, KENAWY A M, et al. Vibriosis outbreaks in farmed Nile Tilapia (Oreochromis niloticus) caused by Vibrio mimicus and V. cholerae [J]. Aquaculture International, 2022, 30(5): 2661−2677. doi: 10.1007/s10499-022-00921-8 [3] SONY M, SUMITHRA T G, ANUSREE V N, et al. Antimicrobial resistance and virulence characteristics of Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio harveyi from natural disease outbreaks of marine/estuarine fishes [J]. Aquaculture, 2021, 539: 736608. doi: 10.1016/j.aquaculture.2021.736608 [4] 朱苏琴, 纪荣兴, 苏永全, 等. 河流弧菌(Vibrio fluvialis)对大黄鱼(Pseudosciaena crocea)鳃黏液黏附特性研究 [J]. 海洋与湖沼, 2012, 43(2):389−393. doi: 10.11693/hyhz201202030030ZHU S Q, JI R X, SU Y Q, et al. Study on adhesion characteristics of Vibrio fluvialis to the gill mucus of Pseudosciaena crocea [J]. Oceanologia et Limnologia Sinica, 2012, 43(2): 389−393. (in Chinese) doi: 10.11693/hyhz201202030030 [5] URBAN-CHMIEL R, MAREK A, STĘPIEŃ-PYŚNIAK D, et al. Antibiotic resistance in bacteria-a review [J]. Antibiotics, 2022, 11(8): 1079. doi: 10.3390/antibiotics11081079 [6] ZHANG W W, LI C H, GUO M. Use of ecofriendly alternatives for the control of bacterial infection in aquaculture of sea cucumber Apostichopus japonicus [J]. Aquaculture, 2021, 545: 737185. doi: 10.1016/j.aquaculture.2021.737185 [7] CAKIR-KOC R. Production of anti-SAG1 IgY antibody against Toxoplasma gondii parasites and evaluation of antibody activity by ELISA method [J]. Parasitology Research, 2016, 115(8): 2947−2952. doi: 10.1007/s00436-016-5047-9 [8] ZHANG L P, LIN L, QIN Z D. A review on the application of chicken immunoglobulin Y in aquaculture [J]. Reviews in Aquaculture, 2024, 16(1): 536−551. doi: 10.1111/raq.12850 [9] EL-KAFRAWY S A, ABBAS A T, OELKRUG C, et al. IgY antibodies: The promising potential to overcome antibiotic resistance [J]. Frontiers in Immunology, 2023, 14: 1065353. doi: 10.3389/fimmu.2023.1065353 [10] PEREIRA E P V, VAN TILBURG M F, FLOREAN E O P T, et al. Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review [J]. International Immunopharmacology, 2019, 73: 293−303. doi: 10.1016/j.intimp.2019.05.015 [11] GADDE U, RATHINAM T, LILLEHOJ H S. Passive immunization with hyperimmune egg-yolk IgY as prophylaxis and therapy for poultry diseases: A review [J]. Animal Health Research Reviews, 2015, 16(2): 163−176. doi: 10.1017/S1466252315000195 [12] HUSSEIN M A, REHAN I F, REHAN A F, et al. Egg yolk IgY: A novel trend of feed additives to limit drugs and to improve poultry meat quality [J]. Frontiers in Veterinary Science, 2020, 7: 350. doi: 10.3389/fvets.2020.00350 [13] REHAN I F, REHAN A F, ABOUELNAGA A F, et al. Impact of dietary egg yolk IgY powder on behavior, meat quality, physiology, and intestinal Escherichia coli colonization of broiler chicks [J]. Frontiers in Veterinary Science, 2022, 9: 783094. doi: 10.3389/fvets.2022.783094 [14] GAO X J, ZHANG X J, SUN J J, et al. Passive protection effect of anti-Vibrio anguillarum IgY-encapsulated feed on half-smooth tongue sole (Cynoglossus semilaevi ) against V. anguillarum [J]. Fish & Shellfish Immunology, 2016, 56: 483−488. [15] THOMSEN K, CHRISTOPHERSEN L, BJARNSHOLT T, et al. Anti-Pseudomonas aeruginosa IgY antibodies augment bacterial clearance in a murine pneumonia model [J]. Journal of Cystic Fibrosis, 2016, 15(2): 171−178. doi: 10.1016/j.jcf.2015.08.002 [16] GUIMARÃES M C C, AMARAL L G, RANGEL L B A, et al. Growth inhibition of Staphylococcus aureus by chicken egg yolk antibodies [J]. Archivum Immunologiae et Therapiae Experimentalis, 2009, 57(5): 377−382. doi: 10.1007/s00005-009-0041-x [17] GAO X J, ZHANG X J, LIN L, et al. Passive Immune-Protection of Litopenaeus vannamei against Vibrio harveyi and Vibrio parahaemolyticus Infections with Anti-Vibrio Egg Yolk (IgY)-Encapsulated Feed [J]. International Journal of Molecular Sciences, 2016, 17(5): 723. doi: 10.3390/ijms17050723 [18] YI L Z, QIN Z D, LIN H Z, et al. Features of chicken egg yolk immunoglobulin (IgY) against the infection of red-spotted grouper nervous necrosis virus [J]. Fish & Shellfish Immunology, 2018, 80: 534−539. [19] 胡青青, 赵肃清, 贺攀, 等. 抗须癣毛癣菌细胞壁蛋白的卵黄抗体制备和鉴定 [J]. 中国免疫学杂志, 2017, 33(9):1350−1354. doi: 10.3969/j.issn.1000-484X.2017.09.015HU Q Q, ZHAO S Q, HE P, et al. Preparation and identification of specific chicken egg yolk immunoglobulins against cell wall protein of Trichophyton mentagrophytes [J]. Chinese Journal of Immunology, 2017, 33(9): 1350−1354. (in Chinese) doi: 10.3969/j.issn.1000-484X.2017.09.015 [20] 翟玥, 曲笑锋, 庞博, 等. 副溶血性弧菌高免卵黄抗体的制备和不同提纯方法效果的比较 [J]. 吉林大学学报(医学版), 2017, 43(2):441−445.ZHAI Y, QU X F, PANG B, et al. Preparation of high immunity yolk antibody against Vibrio parahemolyticus and comparison of effectiveness between different extraction methods [J]. Journal of Jilin University (Medicine Edition), 2017, 43(2): 441−445. (in Chinese) [21] 姜肖军, 郭亚男, 陈秀红, 等. 鸡滑液囊支原体卵黄抗体的制备与鉴定 [J]. 中国兽医科学, 2020, 50(8):1018−1022.JIANG X J, GUO Y N, CHEN X H, et al. The production and identification of eeg yalk antibody(IgY) against Mycoplasma synoviae [J]. Chinese Veterinary Science, 2020, 50(8): 1018−1022. (in Chinese) [22] XU L, XU Y P, HE L Y, et al. Immunomodulatory effects of chicken egg yolk antibodies (IgY) against experimental Shewanella marisflavi AP629 infections in sea cucumbers (Apostichopus japonicus) [J]. Fish & Shellfish Immunology, 2019, 84: 108−119. [23] XU L, CHE J, XU Y P, et al. Oral administration of microencapsulated egg yolk immunoglobulin (IgY) in turbot (Scophthalmus maximus) to combat against Edwardsiella tarda 2CDM001 infections [J]. Fish & Shellfish Immunology, 2020, 106: 609−620. [24] QIN Z D, BABU V S, LI N Q, et al. Protective effects of chicken egg yolk immunoglobulins (IgY) against experimental Aeromonas hydrophila infection in blunt snout bream (Megalobrama amblycephala) [J]. Fish & Shellfish Immunology, 2018, 78: 26−34. [25] 安芳兰, 李菁, 张荣, 等. 卵黄抗体在疾病防治中应用的研究进展 [J]. 安徽农业科学, 2016, 44(5):68−71. doi: 10.3969/j.issn.0517-6611.2016.05.026AN F L, LI J, ZHANG R, et al. Research progress of egg yolk antibody in the prevention and treatment of various diseases [J]. Journal of Anhui Agricultural Sciences, 2016, 44(5): 68−71. (in Chinese) doi: 10.3969/j.issn.0517-6611.2016.05.026 [26] TSUBOKURA K, BERNDTSON E, BOGSTEDT A, et al. Oral administration of antibodies as prophylaxis and therapy in Campylobacter jejuni-infected chickens [J]. Clinical and Experimental Immunology, 1997, 108(3): 451−455. [27] AUZUREEN A M Z, MICHAEL M S, MOHAMED M, et al. Detection of pathogenic Vibrio species and antibiogram activity in Asian Seabass (Lates calcarifer) in Tumpat, Kelantan [J]. Tropical Biomedicine, 2022, 39(4): 569−574. doi: 10.47665/tb.39.4.013 [28] BAO J J, GUO D K, JIN L, et al. Accurate identification of diverse N-acyl homoserine lactones in marine Vibrio fluvialis by UHPLC-MS/MS [J]. Current Microbiology, 2022, 79(6): 181. doi: 10.1007/s00284-022-02879-5 [29] CHEN Y H, AI X H, YANG Y B. Vibrio cholerae: A pathogen shared by human and aquatic animals [J]. The Lancet Microbe, 2022, 3(6): e402. doi: 10.1016/S2666-5247(22)00125-2 [30] XIAO X X, LIN Z Q, HUANG X H, et al. Rapid and sensitive detection of Vibrio vulnificus using CRISPR/Cas12a combined with a recombinase-aided amplification assay [J]. Frontiers in Microbiology, 2021, 12: 767315. doi: 10.3389/fmicb.2021.767315 [31] LIANG J F, PENG C, LI P Y, et al. A review of detection of antibiotic residues in food by surface-enhanced Raman spectroscopy [J]. Bioinorganic Chemistry and Applications, 2021, 2021: 8180154. [32] DOU L N, ZHANG Y J, BAI Y C, et al. Advances in chicken IgY-based immunoassays for the detection of chemical and biological hazards in food samples [J]. Journal of Agricultural and Food Chemistry, 2022, 70(4): 976−991. doi: 10.1021/acs.jafc.1c06750 [33] FISHMAN J B, BERG E A. Isolation of IgY from chicken eggs[J]. Cold Spring Harbor Protocols, 2018, 2018(6): pdb. prot099150. [34] KARACHALIOU C E, VASSILAKOPOULOU V, LIVANIOU E. IgY technology: Methods for developing and evaluating avian immunoglobulins for the in vitro detection of biomolecules [J]. World Journal of Methodology, 2021, 11(5): 243−262. doi: 10.5662/wjm.v11.i5.243 [35] KOVACS-NOLAN J, PHILLIPS M, MINE Y. Advances in the value of eggs and egg components for human health [J]. Journal of Agricultural and Food Chemistry, 2005, 53(22): 8421−8431. doi: 10.1021/jf050964f [36] MINE Y, KOVACS-NOLAN J. Biologically active hen egg components in human health and disease [J]. The Journal of Poultry Science, 2004, 41(1): 1−29. doi: 10.2141/jpsa.41.1 [37] CAI R Z, LIU N, GUO P H, et al. Protective effects of chicken egg yolk immunoglobulins (IgYs) against Vibrio vulnificus infections [J]. Journal of Immunology Research, 2021, 2021: 6678513. [38] RANJBAR M, BEHROUZ B, NOROUZI F, et al. Anti-PcrV IgY antibodies protect against Pseudomonas aeruginosa infection in both acute pneumonia and burn wound models [J]. Molecular Immunology, 2019, 116: 98−105. doi: 10.1016/j.molimm.2019.10.005 [39] SHI H Y, ZHU J, ZOU B Y, et al. Effects of specific egg yolk immunoglobulin on pan-drug-resistant Acinetobacter baumannii [J]. Biomedicine & Pharmacotherapy, 2017, 95: 1734−1742. [40] 张文平, 谢水祥, 傅颖媛, 等. 抗白念珠菌卵黄免疫球蛋白对白念珠菌生长及黏附的影响 [J]. 临床皮肤科杂志, 2005, 34(12):814−815. doi: 10.3969/j.issn.1000-4963.2005.12.009ZHANG W P, XIE S X, FU Y Y, et al. Effect of anti-Candida albicans yolk immunoglobulin on the growth and adhesion of Candida albicans [J]. Journal of Clinical Dermatology, 2005, 34(12): 814−815. (in Chinese) doi: 10.3969/j.issn.1000-4963.2005.12.009 [41] 贺维朝, 张会艳, 王浩, 等. 卵黄抗体提取方法及其在畜禽细菌性肠道疾病防治中的应用 [J]. 中国畜牧兽医, 2021, 48(2):640−649.HE W Z, ZHANG H Y, WANG H, et al. Extraction methods of yolk antibody and its application in prevention and treatment of bacterial intestinal diseases in livestock and poultry [J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(2): 640−649. (in Chinese) [42] 陈翠萍, 杨朝晖, 王永谦. IgY抗体在体外和体内对幽门螺杆菌作用的研究 [J]. 中华微生物学和免疫学杂志, 2002, 22(1):37−40. doi: 10.3760/j:issn:0254-5101.2002.01.013CHEN C P, YANG Z H, WANG Y Q. Neutralization of cytotoxic activity of Helicobacter pylori and treatment of mice by specific IgY Antibody [J]. Chinese Journal of Microbiology and Immunology, 2002, 22(1): 37−40. (in Chinese) doi: 10.3760/j:issn:0254-5101.2002.01.013 [43] KOTA R K, REDDY P N, SREERAMA K. Application of IgY antibodies against staphylococcal protein A (SpA) of Staphylococcus aureus for detection and prophylactic functions [J]. Applied Microbiology and Biotechnology, 2020, 104(21): 9387−9398. doi: 10.1007/s00253-020-10912-5 [44] 姚羽菲, 龙敏, 文荟淋, 等. 抗铜绿假单胞菌IgY的制备优化及抑菌效果 [J]. 检验医学与临床, 2018, 15(2):202−205,208. doi: 10.3969/j.issn.1672-9455.2018.02.018YAO Y F, LONG M, WEN H L, et al. The optimized preparation of IgY against p. aeruginosa and the detection of its antibiotic effect [J]. Laboratory Medicine and Clinic, 2018, 15(2): 202−205,208. (in Chinese) doi: 10.3969/j.issn.1672-9455.2018.02.018 [45] WANG Z B, LI J, LI J Z, et al. Protective effect of chicken egg yolk immunoglobulins (IgY) against enterotoxigenic Escherichia coli K88 adhesion in weaned piglets [J]. BMC Veterinary Research, 2019, 15(1): 234. doi: 10.1186/s12917-019-1958-x [46] KASSIM N, MTENGA A B, SHIM W B, et al. The in vitro and in vivo efficacy of hen IgY against Vibrio parahaemolyticus and Vibrio vulnificus [J]. Journal of Microbiology and Biotechnology, 2012, 22(10): 1423−1431. doi: 10.4014/jmb.1204.04006 [47] ZHANG M X, GENG H J, TARIQ JAVED M, et al. Passive protection of Japanese pufferfish (Takifugu rubripes) against Vibrio harveyi infection using chicken egg yolk immunoglobulins (IgY) [J]. Aquaculture, 2021, 532: 736009. doi: 10.1016/j.aquaculture.2020.736009 -
下载:
点击查看大图
图(6)
计量
- 文章访问数: 45
- HTML全文浏览量: 25
- PDF下载量: 61
- 被引次数: 0
