Preparation and In Vitro Inhibitory Effects of Specific Tripple Anti-vibrio Egg Yolk Antibody
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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
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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)。
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