从灿烂弧菌中克隆和特性分析毒力因子 Hop。
Cloning and characterization of the virulence factor Hop from Vibrio splendidus.
机构信息
State Key Laboratory for Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, PR China.
State Key Laboratory for Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, PR China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, PR China.
出版信息
Microb Pathog. 2020 Feb;139:103900. doi: 10.1016/j.micpath.2019.103900. Epub 2019 Nov 29.
BACKGROUND
Vibrio splendidus is an aquaculture pathogen that can cause skin ulcer syndrome (SUS) in Apostichopus japonicus. HopPmaJ is a type III system effector (T3SE) that has been reported to be an important virulence factor. In this study, a gene named hop, which encodes HopPmaJ in V. splendidus was cloned and its cytotoxicity to coelomocytes and its effects on the expression of immune-related genes in A. japonicus were characterized.
METHODS
Real time reverse transcription PCR (RT-PCR) was used to determine the expression of the hop gene under various conditions. To obtain the purified Hop, hop gene was conditionally expressed in Escherichia coli BL21(DE3) and was purified by GST tag. The cytotoxicity of Hop to coelomocyte was determined using MTT method, and the effect of Hop on the expression of immune-related genes was determined using real time RT-PCR.
RESULTS
The deduced amino acid sequence of Hop from V. splendidus shared 84%-96% homology with those of Hops from other Vibrio spp. The expression of hop gene was induced not only by host-pathogen contact but also by high cell density. Purified recombinant Hop (rHop) showed cytotoxicity to the coelomocyte of A. japonicus. The cell viability decreased to approximately 42%, 26%, 32%, 30% and 20%, when 30, 50, 60, 80 and 100 μL of purified rHop was added, respectively. After being injected with rHop, the expression levels of immune-related genes that encode complement component (C1q) and caspase were significantly increased, and the production of reactive oxygen species were also increased in A. japonicus.
CONCLUSION
Our results indicated that Hop not only contributed to the cytotoxicity to coelomocyte, but also caused immune response in A. japonicus.
背景
灿烂弧菌是一种水产养殖病原体，可引起刺参的皮肤溃疡综合征（SUS）。HopPmaJ 是一种 III 型系统效应物（T3SE），已被报道为重要的毒力因子。在本研究中，克隆了灿烂弧菌中编码 HopPmaJ 的基因 hop，并研究了其对刺参血细胞的细胞毒性及其对刺参免疫相关基因表达的影响。
方法
采用实时 RT-PCR 测定不同条件下 hop 基因的表达。通过 GST 标签，在大肠杆菌 BL21(DE3) 中条件表达 hop 基因，获得纯化的 Hop。采用 MTT 法测定 Hop 对血细胞的细胞毒性，采用实时 RT-PCR 法测定 Hop 对免疫相关基因表达的影响。
结果
从灿烂弧菌中推导的 Hop 氨基酸序列与其他弧菌属的 Hops 同源性为 84%-96%。宿主-病原体接触不仅诱导 hop 基因的表达，高密度细胞也能诱导 hop 基因的表达。纯化的重组 Hop（rHop）对刺参血细胞具有细胞毒性。当加入 30、50、60、80 和 100 μL 的纯化 rHop 时，细胞活力分别降低到约 42%、26%、32%、30%和 20%。注射 rHop 后，编码补体成分（C1q）和半胱天冬酶的免疫相关基因的表达水平显著增加，刺参的活性氧产生也增加。
结论
我们的结果表明，Hop 不仅有助于血细胞的细胞毒性，而且还引起刺参的免疫反应。
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