蛙类脑膜脓毒性伊丽莎白菌多克隆抗体的制备及应用

张新艳; 曾占壮; 叶小军; 卓玉琛; 钟全福; 余培建

doi:10.19303/j.issn.1008-0384.2021.07.008

蛙类脑膜脓毒性伊丽莎白菌多克隆抗体的制备及应用

doi: 10.19303/j.issn.1008-0384.2021.07.008

福建省淡水水产研究所，福建　福州　350002

基金项目: 福建省科技计划公益类专项（2019R1014-5）；福建省海洋与渔业结构调整专项（闽财指[2017]1177号、闽财指[2018]829号）

详细信息

作者简介:
张新艳（1980−），女，硕士，高级工程师，研究方向：水产养殖动物病害（E-mail：swallowz47@163.com）

通讯作者:
曾占壮（1972−），男，硕士，副研究员，研究方向：水产养殖动物病害（E-mail：zengzhanzhuang@126.com）

中图分类号: S 947
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出版历程
- 收稿日期: 2021-04-15
- 修回日期: 2021-05-31
- 网络出版日期: 2021-07-13
- 刊出日期: 2021-07-28

Preparation and Application of Rabbit Polyclonal Antibodies against Elizabethkingia meningosepticum on Frogs

The Freshwater Fisheries Research Institute of Fujian Province, Fuzhou, Fujian　350002, China

摘要

摘要: 目的制备蛙类脑膜脓毒性伊丽莎白菌多克隆抗体，建立间接ELISA检测方法，进行病原菌在宿主体内的示踪研究，为病原菌的致病机理研究奠定基础。方法将分离自患歪头病的棘胸蛙的脑膜脓毒性伊丽莎白菌（Elizabethkingia meningosepticum）RsB1151018NA甲醛灭活后，免疫新西兰大白兔制备兔抗血清。通过优化反应条件，建立脑膜脓毒性伊丽莎白菌间接ELISA的检测方法；兔抗血清多克隆抗体采用Protein A亲和层析柱分离纯化，以异硫氰酸荧光素（FITC）标记，标记后的抗体与感染RsB1151018NA的棘胸蛙不同脏器组织的冷冻切片反应，荧光显微镜观察，跟踪RsB1151018NA在棘胸蛙体内的迁移。结果试验结果表明，制备的兔抗棘胸蛙脑膜脓毒性伊丽莎白菌多克隆抗体血清效价为1∶5.12×10⁵，纯化后的抗体效价为1∶1.6×10⁴，且建立的间接ELISA方法检测脑膜脓毒性伊丽莎白菌具有高度特异性，与其他水产常见病原菌没有交叉反应，检测灵敏度为1.0×10⁴ CFU·mL⁻¹。脑膜脓毒性伊丽莎白菌在棘胸蛙体内随血液循环而分布至全身，可侵袭肌肉、肝脏、肾脏、脾脏、肠道、心脏、眼和脑等组织器官，肾脏、眼和脑感染程度最严重，推测这三个组织器官是其靶器官；创伤感染是可能的感染途径之一。结论建立的检测方法能够快速、灵敏、特异地检测脑膜脓毒性伊丽莎白菌，对蛙类歪头病的防控和早期诊断可提供理论参考依据；另外利用免疫荧光检测病原菌在宿主体内的迁移，研究病原的致病机理及发病过程等，能为防治药物的研发和防治方法的确定提供理论依据。
- 脑膜脓毒性伊丽莎白菌 /
- 多克隆抗体 /
- 酶联免疫反应 /
- 检测 /
- 蛙“歪头病”
Abstract: Objective Rabbit polyclonal antibodies against Elizabethkingia meningosepticum (Em) were prepared to establish an indirect ELISA detection to trace the pathogenic attack in organs of frogs (Rana spp). Method The strain RsB1151018NA of Em from a diseased R. spinosa was isolated, formalin-inactivated, and injected into a rabbit for the serum preparation. An optimized indirect ELISA using the rabbit polyclonal antibodies was developed for tracing RsB1151018NA. The rabbit immunoglobulin G was purified on a protein A column. The purified antibodies were marked with FITC and further purified on a desalting column Sephadex G-25 for subsequently tracing the pathogen in organs of the inoculated frogs. Results The obtained rabbit polyclonal antibodies showed a titer up to 1 ∶ 5.12×10⁵, and 1 ∶ 1.6×10⁴ after purification. The indirect ELISA assay on the polyclonal antibody was specific and sensitive in detecting Em without cross-reaction on other bacteria. It had a detection limit of 1.0×10⁴ cfu·mL⁻¹. According to the in vivo tracing test, RsB1151018NA distributed throughout the entire frog body by blood circulation with the most serious infections in the kidney, eyes, and brain. Conclusion The optimized indirect ELISA detection of Em was rapid, sensitive, and specific in tracing the bacterial infection on R. spp. It was considered applicable for early diagnosis of the “crooked-head disease” and breeding of healthy frogs for food and medicine.
- Elizabethkingia meningosepticum /
- polyclonal antibodies /
- ELISA /
- detection /
- “crooked-head disease” of frogs

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图 1 兔抗血清亲和层析纯化图谱

Figure 1. Chromatography of polyclonal antibody purified by protein A affinity method

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图 2 兔抗血清纯化效果分析

注：1：蛋白marker 2：纯化抗体。

Figure 2. SDS-PAGE analysis on purified polyclonal antibody

Note: Lane l: protein marker; Lane 2: purified polyclonal antibodies.

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图 3 Sephadex G-25脱盐柱纯化FITC标记抗体

Figure 3. Purification of IgG-FITC-marked antibodies with desalting column Sephadex G-25

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图 4 RsB1151018NA体内示踪

Figure 4. In vivo tracing of RsB1151018NA in frog

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图 5 感染24 h肝切片

Figure 5. Fluorescence microscopic detection on liver infection in 24 h

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表 1 用于抗体特异性测定的菌株

Table 1. Bacterium used for ELISA detection

菌株 Strain	细菌种类 Bacterial species
LCCiL90625NA-C	脑膜脓毒性伊丽莎白菌 E. meningosepticum
ATCC13525	荧光假单胞菌 Pesudomonas fluorescens
ATCC9027	铜绿假单胞菌 P. aeruginosa
ML316	嗜水气单胞菌 Aeromonas hydrophila
Ah0081	嗜水气单胞菌 A. hydrophila
Ac60324	豚鼠气单胞菌 A. caviae
96-5	温和气单胞菌 A. sobria
Fp60325NA	温和气单胞菌 A. sobria
LCCiL90625NA	类志贺邻单胞菌 Plesiomonas shigelloides
ATCC17802	副溶血弧菌 Vibrio parahaemolyticus
FJ04-L1	创伤弧菌 V. vulnificus
Js60517NA1	溶藻弧菌 V. alginolyticus
AL60306NA1	迟缓爱德华氏菌 Edwardsiella tarda

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表 2 兔抗多克隆抗体血清效价分析

Table 2. Titer of polyclonal antibody against Em

样品 Samples	OD₄₅₀	结果判定 Result
空白血清	0.1919	NC
1∶500	2.968	+
1∶2000	2.907	+
1∶8000	2.889	+
1∶32000	2.885	+
1∶128000	2.547	+
1∶512000	1.201	+
1∶2048000	0.36	−
注：NC，阴性对照，“+”表示阳性，“−”表示阴性，S/N≥2.1为阳性。 Note：“NC”indicates negative control, “+” indicates positive,“−” indicates negative, S/N≥2.1 represent for positive.

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表 3 兔多抗检测灵敏度分析

Table 3. Sensitivity of indirect ELISA assay in detecting RsB1151018NA

包被含量 Coating concentration/CFU·mL⁻¹	P/N	结果判定 Result
阴性对照	1	−
1×10⁸	42.1	+
1×10⁷	18.2	+
1×10⁶	7.58	+
1×10⁵	5.06	+
1×10⁴	3.38	+
1×10³	1.66	−
注：“+”表示阳性，“−”表示阴性。 Note: +: positive; −: negative.

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表 4 纯化后抗体效价分析测定

Table 4. Titer of purified polyclonal antibody against Em

样品 Samples	OD₄₅₀	结果判定 Result
1︰500	2.779	+
1︰1 000	2.284	+
1︰2 000	1.963	+
1︰4 000	1.482	+
1︰8 000	1.067	+
1︰16 000	0.643	+
1︰32 000	0.415	−
空白血清	0.268	NC
注：NC，阴性对照，“+”表示阳性，“−”表示阴性，S/N≥2.1为阳性。 Note: NC: negative control; +: positive; −: negative; S/N≥2.1 indicates positive.

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表 5 多克隆抗体检测特异性分析

Table 5. Specificity of polyclonal antibody against RsB1151018NA

菌株 Strain	细菌种类 Bacterial species	OD_{450 nm}	判定结果 Result
RsB11518018NA	脑膜脓毒性伊丽莎白菌 E.meningosepticum	0.362	+
LCCiL90625NA-C	脑膜脓毒性伊丽莎白菌 E. meningosepticum	0.335	+
ATCC13525	荧光假单胞菌 Pesudomonas fluorescens	0.197	−
ATCC9027	铜绿假单胞菌 P. aeruginosa	0.192	−
ML316	嗜水气单胞菌Aeromonas hydrophila	0.186	−
Ah0081	嗜水气单胞菌 A. hydrophila	0.185	−
Ac60324	豚鼠气单胞菌 A. caviae	0.187	−
96-5	温和气单胞菌 A. sobria	0.190	−
Fp60325NA	温和气单胞菌 A. sobria	0.193	−
LCCiL90625NA	类志贺邻单胞菌 Plesiomonas shigelloides	0.187	−
ATCC17802	副溶血弧菌 Vibrio parahaemolyticus	0.194	−
FJ04-L1	创伤弧菌 V. vulnificus	0.189	−
Js60517NA1	溶藻弧菌 V. alginolyticus	0.195	−
AL60306NA1	迟缓爱德华氏菌 Edwardsiella tarda	0.208	−
阴性对照	5% 脱脂奶粉 5%dried separated milk	0.106	NC
注：“+”表示阳性，“−”表示阴性 Note: +: positive; −: negative.

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表 6 FITC标记抗体效价测定

Table 6. Titer of purified IgG-FITC antibodies against Em

样品 Samples	OD₄₅₀	结果判定 Result
1∶50	2.287	+
1∶100	1.906	+
1∶200	1.617	+
1∶400	1.225	+
1∶800	0.781	+
1∶1600	0.567	+
1∶3200	0.342	−
空白血清 Blank serum	0.191	NC
注：NC，阴性对照，“+”表示阳性，“−”表示阴性，S/N≥2.1为阳性。 Note: NC: negative control; +: positive; −: negative; S/N≥2.1 indicates positive.

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表 7 RsB1151018NA体内示踪结果

Table 7. In vivo tracing of RsB1151018NA in frog

时间 Time/h	菌数对数（lgN）
时间 Time/h	肌肉 Muscle	血液 Blood	肝脏 Liver	肾脏 Kidney	脾脏 Spleen	心脏 Heart	肠道 Intestinal	眼 Eye	脑 Brain
6	1.18	0.89	0.41	0.62	0.00	0.66	0.00	0.00	0.00
12	1.08	0.85	0.51	0.93	0.26	0.64	0.00	0.00	0.00
24	0.92	0.83	0.68	1.20	0.41	0.56	0.56	0.41	0.56
36	0.83	0.91	0.75	1.23	0.53	0.45	0.64	0.58	0.83
48	0.41	0.88	0.81	1.16	0.51	0.30	0.51	0.68	0.92
60	0.00	0.83	0.53	0.82	0.45	0.48	0.48	0.72	0.89
72	0.00	0.81	0.30	0.38	0.00	0.00	0.48	0.78	0.90

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