罗非鱼无乳链球菌ΔessA、ΔessB、ΔessC敲除菌株的构建及其特性分析

马艳平; 郝乐; 冯国清; 梁志凌; 马江耀; 柯浩; 刘振兴

doi:10.12131/20210246

罗非鱼无乳链球菌ΔessA、ΔessB、ΔessC敲除菌株的构建及其特性分析

doi: 10.12131/20210246

广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站，广东广州 510640

基金项目: 广东省自然科学基金项目 (2021A1515010498)；广东省农业科学院水产研究中心课题 (XT202232)；佛山市财政专项资金—2021年度共建广东农业科技示范市项目

详细信息

作者简介:
马艳平 (1984—)，女，副研究员，博士，从事水产病害与防控研究。 E-mail: mayanping@gdaas.cn

通讯作者:
刘振兴 (1981—)，男，副研究员，博士，从事水产病害与防控研究。 E-mail: liuzhenxing@gdaas.cn

中图分类号: S 943.116.42
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出版历程
- 收稿日期: 2021-08-26
- 修回日期: 2021-12-14
- 录用日期: 2021-12-21
- 网络出版日期: 2022-02-07
- 刊出日期: 2022-08-05

Construction and characterization research of essA, essB and essC-deleted mutants in Streptococcus agalactiae from tilapia

Institute of Animal Health, Guangdong Academy of Agricultural Sciences/Key Laboratory of Livestock Disease Prevention of Guangdong Province/Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, China

摘要

摘要: VII型分泌系统存在于无乳链球菌 (Streptococcus agalactiae) 中，其组成膜蛋白对底物蛋白的分泌转运至关重要。为此利用热敏型自杀质粒，构建了含氯霉素筛选标签的膜蛋白重组敲除载体pSET4s-ΔessA、pSET4s-ΔessB、pSET4s-ΔessC，经同源重组后，利用PCR技术筛选到了其缺失突变株-无乳链球菌ΔessA、ΔessB、ΔessC。通过菌株生长曲线分析，ΔessA、ΔessB、ΔessC敲除株均较野生株 (WT) 生长过程变慢，其中ΔessA、ΔessB敲除株在生长早期有显著性差异 (P<0.01)。对ESAT6蛋白分泌影响分析显示，与WT株相比，ΔessA、ΔessB、ΔessC敲除株ESAT6的表达量均显著下调 (P<0.01)。结果显示，ΔessA、ΔessB、ΔessC敲除株均较WT株毒力显著降低 (P<0.01)。研究表明，essA、essB、essC为无乳链球菌VII型分泌系统重要的膜蛋白，其基因缺失造成了分泌蛋白ESAT6 mRNA表达水平下降，影响了菌株毒力。
- 罗非鱼 /
- 无乳链球菌 /
- VII型分泌系统膜蛋白 /
- 敲除菌株构建 /
- 生物学特性分析
Abstract: The membrane proteins are critical to substrate protein secretion in VII secretion system existing in Streptococcus agalactiae. In this study, we constructed the membrane proteins knockout vectors with chloramphenicol tag including pSET4s-ΔessA, pSET4s-ΔessB and pSET4s-ΔessC, and after the homologous recombination, we screened the mutant S. agalactiae ΔessA, ΔessB and ΔessC via PCR assays. Based on the growth curve analysis, S. agalactiae ΔessA, ΔessB and ΔessC strains showed significantly slower growth process than the wild type strain (WT). ΔessA and ΔessB strains had significant difference in early stage of growth compared with WT strain (P<0.01). According to the secretion product analysis of ESAT6 protein, S. agalactiae ΔessA, ΔessB and ΔessC strains showed significantly lower ESAT6 product than WT (P<0.01). According to challenge test analysis, the deletion of essA, essB or essC gene significantly reduced virulence of S. agalactiae ΔessA, ΔessB and ΔessC compared with WT (P<0.01). The results suggest that essA, essB and essC proteins are important component membrane proteins of VII secretion system in S. agalactiae, and these genes deletions cause ESAT6 mRNA expression and virulence decline.
- Oreochromis sp. /
- Streptococcus agalactiae /
- Membrane proteins of VII secretion system /
- Knockout strains construction /
- Biological characteristic analysis

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图 1 essA、essB、essC同源臂融合PCR结果

Figure 1. Overlap extension PCR results of essA, essB, essC homologous arms

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图 2 敲除株ΔessA、ΔessB、ΔessC DNA水平PCR鉴定结果

Figure 2. PCR identification results of ΔessA, ΔessB, ΔessC knockout strains at DNA level

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图 3 敲除株ΔessA、ΔessB、ΔessC RNA水平PCR鉴定结果

Figure 3. PCR identification of ΔessA, ΔessB and ΔessC knockout strains at RNA level

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图 4 敲除菌株ΔessA、ΔessB、ΔessC 生长曲线测定结果

Figure 4. Growth curve of ΔessA, ΔessB and ΔessC knockout strains

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图 5 ESAT6 qPCR检测方法的建立

注：a. 扩增效率分析结果；b. 熔解曲线分析结果； c. 扩增产物琼脂糖凝胶检测结果。

Figure 5. qPCR detection method of ESAT6 gene

Note: a. Amplification efficiency analysis result; b. Melt curve analysis result; c. Amplification product detection result based on agarose gel.

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图 6 敲除株ΔessA、ΔessB、ΔessC 与野生株对底物蛋白ESAT6表达量的影响结果

注：**. 差异显著 (P<0.01)。

Figure 6. Effect of substrate protein ESAT6 expression by knockout strains ΔessA, ΔessB, ΔessC and WT strain

Note: **. Significant difference (P<0.01).

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图 7 敲除菌株ΔessA、ΔessB、ΔessC 与野生株毒力试验结果

Figure 7. Virulence test of ΔessA, ΔessB, ΔessC knockout strains and WT strain

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图 8 攻毒死亡鱼解剖及其菌株鉴定结果

注：a. 攻毒死亡鱼解剖症状；b. 攻毒分离菌株革兰氏染色结果；c. 攻毒分离菌株 PCR 鉴定结果。

Figure 8. Anatomical symptom result and identification of strains of infected dead tilapia

Note: a. Anatomical symptom result of infected dead tilapia; b. Gram staining result of isolated strain after infection; c. PCR identification of isolated strain after infection.

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表 1 试验用引物

Table 1. Primers in this study

引物名称 Primer name	引物序列 Primer sequence 5'−3'
重组质粒构建用引物 Primers for recombinant plasmid construction
essAup-FBamHI	5'-GCGGATCCGCCGTAGGCACAGTAGCGACT-3' BamHI
essAup-R	5'-GAGTGAGACTTTAGATTGACGG-3'
essAdown-F	5'-ACGTTGAGCCTCGGAACCCATCGAATTAGAATACACATATTAACATTACT-3'
essAdown-REcoRI	5'-GCGAATTCGATAGAGCGTGCGCTTCTGTCTG-3' EcoRI
essBup-FBamHI	5'-GCGGATCCGGACCAGATTTTTGGCAGTTATC-3' BamHI
essBup-R	5'-CAGCCTGAATAGTTTCTGCATGAT-3'
essBdown-F	5'-GACGTTGAGCCTCGGAACCCATCGAATTATAAAGTTGATTATAATCAAGTGA-3'
essBdown-REcoRI	5'-GCGAATTCAGAAGATGTACTTGTTGTAGTAC-3' EcoRI
essCup-FSalI	5'-GCGTCGACAAACGGCAAGTCGCCAATACCAG-3' SalI
essCup-R	5'-TGCTGACTCCAATCATCACTAG-3'
essCdown-F	5'-GACGTTGAGCCTCGGAACCCATCGAATTACAGGAAATGGCTGATACTTATCAC-3'
essCdown-REcoRI	5'-GCGAATTCTTAACTTTTCATCAGTTACAAT-3' EcoRI
Cat(essA)-F	5'-ATGAAAACCGTCAATCTAAAGTCTCACTCCACCGAACTAGAGCTTGATG-3'
Cat(essB)-F	5'-TATCATGCAGAAACTATTCAGGCTGCACCGAACTAGAGCTTGATGAAAA-3'
Cat(essC)-F	5'-CCTCCTCTAGTGATGATTGGAGTCAGCACACCGAACTAGAGCTTGATGAA-3'
Cat-R	5'-TAATTCGATGGGTTCCGAGGCTC-3'
基因缺失突变株筛选所用引物 Primers for gene deletion mutant strains screening
essA验证-F	5'-GCCGTAGGCACAGTAGCGACTG-3'
essA验证-R	5'-ACATTCAAGGCTAATCGTAA-3'
essB验证-F	5'-TGGAAACAGATTCGTTTGTA-3'
essB验证-R	5'-TACCTACTTTTAGTTTTAG-3'
essC验证-F	5'-CGTCCTCGTGGTATCTATATC-3'
essC验证-R	5'-CAATATCATCCTGACCACGTAAG-3'
essA-F	5'-ATGAAGTTGAAACGATTTTTAG-3'
essA-R	5'-TTAATATGTGTATTCATCCT-3'
essB-F	5'-TGGAAACAGATTCGTTTGTA-3'
essB-R	5'-TACCTACTTTTAGTTTTAGA-3'
essC-F	5'-TCGTGGTATCTATATCATTGCAAC-3'
essC-R	5'-GATAAACAATATCATCCTGACCAC-3'
Real-time PCR所用引物 Primers for real-time PCR
ESAT6-F	5'-ATACTGCTGGTTCTCAACAA-3'
ESAT6-R	5'-GTCAATAACTGCTTGCTCTT-3'
16S-rRNA-F	5'-CGACGATACATAGCCGACC-3'
16S-rRNA-R	5'-CCGTCACTTGGTAGATTTTCC-3'
注：下划线为限制性内切酶位点。 Note: The underlined are restriction endonuclease sites.

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