嗜水气单胞菌分泌蛋白重组疫苗对斑马鱼的免疫保护效力评价

林潇可; 汪玉倩; 林美珍; 黄东平; 苗宇轩; 林向民

doi:10.12131/20220266

嗜水气单胞菌分泌蛋白重组疫苗对斑马鱼的免疫保护效力评价

doi: 10.12131/20220266

林潇可^1,,
汪玉倩²,
林美珍^{1, 3},
黄东平^{1, 3},
苗宇轩^{1, 3},
林向民^{1, 3, ,}

1.
福建农林大学生命科学学院，福建福州 350002
2.
安徽科技学院农学院，安徽滁州 233100
3.
福建农林大学/福建省农业生态过程与安全监控重点实验室，福建福州 350002

基金项目: 福建大学校级自然科学基金(111ZC9051)

详细信息

作者简介:
林潇可 (2001—)，男，本科生，研究方向为微生物分子生态学。E-mail: 937680443@qq.com

通讯作者:
林向民 (1977—)，男，教授，博士，研究方向为微生物分子生态学。E-mail: xiangmin@fafu.edu.cn

中图分类号: S 941.42
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- 文章访问数: 88
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- 被引次数: 0
出版历程
- 收稿日期: 2022-10-06
- 修回日期: 2023-02-15
- 录用日期: 2023-03-21
- 网络出版日期: 2023-03-27

Immune protective efficacy of recombinant vaccine against Aeromonas hydrophila secreted proteins on zebrafish

LIN Xiaoke^1
,,
WANG Yuqian²,
LIN Meizhen^{1, 3},
HUANG Dongping^{1, 3},
MIAO Yuxuan^{1, 3},
LIN Xiangmin^{1, 3
, ,}

1.
School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2.
College of Agriculture, Anhui Science and Technology University, Chuzhou 233100, China
3.
Fujian Agriculture and Forestry University/Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fuzhou 350002, China

摘要

摘要: 嗜水气单胞菌 (Aeromonas hydrophila) 是一种常见的水产致病菌，可引发人畜鱼共患疾病，对水产养殖业造成严重危害，因此预防其传播和感染是当前亟待解决的问题。为开发高效的潜在亚单位疫苗，并为防治嗜水气单胞菌病害提供理论依据，基于前期的研究结果，选取在嗜水气单胞菌强毒株LP-2中表达丰度较高的8种分泌蛋白 (ORF0322、ORF3982、ORF2874、ORF1767、ORF3984、ORF2546、ORF0472、ORF1609) 作为研究目标，首先利用实时荧光定量PCR (qPCR) 技术对其进行基因表达检测，结果发现这8种分泌蛋白在细胞中均可正常表达；然后将这些蛋白进行基因克隆与表达纯化，并将纯化的重组蛋白免疫斑马鱼 (Danio rerio)，连续培养28 d后，发现斑马鱼中相关免疫基因均发生上调，说明这些分泌蛋白可引起宿主发生免疫反应；最后，对这8种分泌蛋白进行免疫保护评价，细菌攻毒实验结果显示，其中6种分泌蛋白 (ORF3982、ORF2874、ORF1767、ORF3984、ORF0472、ORF1609) 的相对免疫保护率 (Relative immune protection rate, RPS) 大于50%，可作为候选疫苗开发。
- 嗜水气单胞菌 /
- 分泌蛋白 /
- 斑马鱼 /
- 疫苗 /
- 免疫保护性 /
- 蛋白重组
Abstract: Aeromonas hydrophila is a prevalent aquatic pathogen that poses a risk to fishery production and can cause zoonotic diseases. Therefore, prevention of transmission and infection of A. hydrophila is a critical issue to address. To develop a potential subunit vaccine and provide a theoretical basis for better preventing and treating A. hydrophila disease, we selected eight secreted proteins (ORF0322, ORF3982, ORF2874, ORF1767, ORF3984, ORF2546, ORF0472 and ORF1609) from an A. hydrophila virulent strain LP-2 based on our previous research. The qPCR test shows that all selected proteins were expressed normally, then they were cloned and purified. The cloned and purified recombinant proteins were administered to zebrafish through injection. After 28 days of immunization, qPCR analysis detects increasing expression of all relevant immune genes in the zebrafish, suggesting that these recombinant proteins were capable of eliciting an immune response. In order to evaluate the immune protection offered by the eight selected secreted proteins, a bacterial challenge experiment was conducted. The results show that six of the secreted proteins (ORF3982, ORF2874, ORF1767, ORF3984, ORF0472, ORF1609) exhibited a relative immune protection rate (RPS) greater than 50%, indicating that they held promise as vaccine candidates against A. hydrophila infection.
- Aeromonas hydrophila /
- Secreted protein /
- Danio rerio /
- Vaccine /
- Immunoprotection /
- Protein recombination

HTML全文

图 1 8种分泌蛋白在嗜水气单胞菌LP-2菌株中的mRNA 表达结果

Figure 1. mRNA expression results of eight excreted proteins in LP-2 strain

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图 2 8个目的基因PCR扩增结果

Figure 2. PCR amplification results of eight target genes

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图 3 高表达重组菌株菌液PCR验证结果

Figure 3. PCR verification results of recombinant strain with high expression

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图 4 纯化蛋白SDS-PAGE 结果

Figure 4. SDS-PAGE results of purified protein

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图 5 斑马鱼的免疫相关基因表达结果

注：*. P<0.05；**. P<0.01；***. P<0.005。

Figure 5. Expression results of immune-related genes in zebrafish

Note: *. P<0.05；**. P<0.01；***. P<0.005。

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图 6 攻毒后注射免疫下接种的斑马鱼的累积成活率及相对免疫保护率

Figure 6. Cumulative survival rate and relative immune protection rate (RPS) of zebrafish inoculated with injection immunization after challenge

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表 1 本研究中选取的分泌蛋白及引物序列

Table 1. Secreted proteins and their primer pairs used in this study

序号 No.	基因 Gene	蛋白质 Protein	核苷酸长度 Nucleotide length/bp	蛋白描述 Protein description	引物序列 (5'—3') Primer sequence (5'−3')
1	orf0322	ORF0322	1 065	假定蛋白 Hypothetical protein	F: GGGGAATTCATGAGGAATCAGGCTGTCATTTTGT R: CCCAAGCTTTCAGGGCCTGGTGGGGGCG
2	orf3982	ORF3982	543	P型菌毛蛋白 PapA	F: AAAGAATTCATGAAAGCTAACAAGAATCTGGTTG R: CCCAAGCTTTTACTCGTAGGAGAGAGTAAAGTTG
3	orf2874	ORF2874	1 866	溶细胞素和溶血素、溶血素、孔道形成蛋白 Cytolysin and hemolysin, HlyA, Pore forming	F: GGGGAATTCATGAAAAACAAAAAACCACGCAAAT R: CCCAAGCTTTCAGTGACTGGCCGGTGGC
4	orf1767	ORF1767	1 482	假定蛋白 Hypothetical protein	F: AAAGAATTCATGAAGACTTTTAAGGTTTCAATTA R: AAAAGGCTTTTAGCGGTTGACGTATTCCTCAA
5	orf3984	ORF3984	2 748	微生物胶原酶 Microbial collagenase	F: AAAGAATTCATGAACAATCTGGGTACCAGGTT R: AAAAGGCTTTCAGTGGGAGGAGTCGTTGG
6	orf2546	ORF2546	519	宿主细胞蛋白 Hcp	F: CCCGAATTCATGCCAACTCCATGTTATATCAGCA R: GGGAAGCTTTTACGCCTCGATCGGCGCA
7	orf0472	ORF0472	930	溶血素 Hemolysin	F: GGGGAATTCATGTTTGGCGACAGCCTCTCC R: CCCAAGCTTTCAGTGGGCGAGGAACTCGTAT
8	orf1609	ORF1609	1 875	假定细胞外丝氨酸蛋白酶 Putative extracellular serine protease	F: GGGGAATTCATGAGAAAAACATCGTTAGCGTTGG R: CCCAAGCTTTCATGAGCGGGCGGCATCG

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表 2 免疫相关基因表达的引物

Table 2. Primers for expression of immune-related genes

序号 No.	基因 Gene	引物序列 (5'—3') Primer sequence (5'−3')
1	Lyz	F: GATTTGAGGGATTCTCCATTGG R: CCGTAGTCCTTCCCCGTATCA
2	IL1β	F: TGGACTTCGCAGCACAAAATG R: GTTCACTTCACGCTCTTGGATC
3	MHC I	F: GGAGTTCACCTTGCTTATGC R: CCCTCTGACCCATTCTTGT
4	MHC II	F: TGACTCAACTGTCCGTGATA R: CCATTAGCCATCTCCATAGTG
5	IL8	F: GTCGCTGCATTGAAACAGAA R: CTTAACCCATGGAGCAGACC
6	IL10	F: TCACGTCATGAACGAGATCC R: CCTCTTGCATTTCACCATATCC
7	IL15	F: ACAGAGGAAGAAGCCTACAG R: GCGATGAAGACGAGAAAGAC

下载: 导出CSV

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