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Molecular Cloning of sodB Gene from vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

Asian Agricultural Research 2024年第7期16卷 42-47页

作者： Shuai YANG Yingying JIANG Haiyun FENG Weijie ZHANG Na WANG Xiaonan LU Huanying PANG Fisheries College Guangdong Ocean UniversityZhanjiang 524025China Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture&Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes Zhanjiang 524025China Chinese Academy of Inspection and Quarantine Beijing100176China Department of Food Science and Agricultural Chemistry Faculty of Agricultural and Environmental SciencesMcGill UniversitySainte-Anne-de-Bellevue H9X 3V9Canada

vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of vibrio alginolyticus HY9901 in order to amplify the full length of the gene by *** results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino *** predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of *** prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane *** amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal *** probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the *** amino acid sequence of the sodB gene of *** exhibited 98%-100%homology to other vibrio species,clustering into the same subfamily with ***,indicating a relatively close relationship between *** the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,*** similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.

关键词： vibrio alginolyticus Gene cloning sodB gene Bioinformatics analysis

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Molecular Cloning and Bioinformatics Analysis of msrA Gene from vibrio alginolyticus Strain HY9901

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Agricultural Biotechnology 2024年第4期13卷 1-5,10页

作者： Zhiqing WEI Zhihang CHEN Yingzhu WEI Na WANG Huanying PANG Fisheries College Guangdong Ocean UniversityZhanjiang 524025China Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture&Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes Zhanjiang 524025China Chinese Academy of Inspection and Quarantine Beijing 100176China

[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 *** protein had a stable structure,and it was hydrophobic *** structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane *** amino acid sequence of MsrA contained multiple signal binding *** subcellular localization showed that MsrA protein was most likely located in the *** analysis showed that MsrA of *** had high homology with other vibrio species,and the highest homology with *** the prediction of functional domains,MsrA had the function of methionine sulfoxide *** secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the *** similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.

关键词： vibrio alginolyticus Gene cloning MsrA Bioinformatics analysis

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Molecular Cloning and Bioinformatics Analysis of cyaA Gene of vibrio alginolyticus Strain HY9901

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Agricultural Biotechnology 2024年第4期13卷 6-10页

作者： Shi WANG Peng ZHOU Yujia ZHANG Weijie ZHANG Na WANG Huanying PANG Fisheries College Guangdong Ocean UniversityZhanjiang 524025China Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture&Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes Zhanjiang 524025China Chinese Academy of Inspection and Quarantine Beijing 100176China

[Objectives]This study was conducted to explore the biological functions of cyaA gene of vibrio alginolyticus.[Methods]With DNA of *** HY 9901 as a template,primers were designed according to the sequence of cyaA gene,and the cyaA gene was amplified by *** analysis was performed.[Results]The cyaA gene of *** HY9901 was 2529 bp in size,and encoded 842 amino *** molecular structure of CyaA protein was C_(4358)H_(6745)N_(1171)O_(1286)S_(35).Its theoretical molecular weight was 97.24167 kDa and the theoretical pI value was *** had no signal peptide and transmembrane *** protein had three N-terminal glycosylation sites,one cAMP and cGMP-dependent protein kinase phosphorylation site,nine protein kinase C phosphorylation sites,nine casein kinase II phosphorylation sites,one tyrosine kinase phosphorylation site,seven N-terminal myristoylation sites,one pentenyl binding site and ten microbody C-terminal localization signal *** localization prediction showed that CyaA protein was mainly located in the nucleus and *** multi-sequence alignment and phylogenetic tree construction,it was concluded that *** had high CyaA homology with other vibrio *** of *** was clustered with vibrio fluminensis and vibrio marinisedimini,and they were closely *** secondary structure of CyaA protein consisted ofα-helixes(43.11%),random coils(38.00%)and extended strands(14.49%).In protein network interaction,it was found that the proteins adjacent to CyaA protein were Crp-2,CpdA,Crr,PtsG-2,ANP67209.1,Crp-1,PykF,Pyk,RelA and Ndk.[Conclusions]This study provides a new idea for formulating strategies for the prevention and control of vibriosis.

关键词： vibrio alginolyticus cyaA gene Bioinformatics analysis

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Disruption of bacterial balance in the gut of Portunus trituberculatus induced by vibrio alginolyticus infection

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Journal of Oceanology and Limnology 2018年第5期36卷 1891-1898页

作者： XIA Mengjie PEI Feng MU Changkao YE Yangfang WANG Chunlin Key Laboratory of Applied Marine Biotechnology（Ningbo University） Ministry of EducationNingbo 315211China Collaborative Innovation Center for Zhejiang Marine High-Efficiency and Healthy Aquaculture Ningbo 315211China

A bstract Gut microbiota impacts the health of crustaceans. V ibrio alginolyticus is a main causative pathogen that induces the vibriosis in farmed swimming crabs, Portunus trituberculatus. However, it remains unknown whether gut bacteria perform functions during the progression of vibriosis. In this study, 16 SrRNA gene amplicon sequencing was used to investigate temporal alteration of gut bacterial community in swimming crabs in response to 72-h V. alginolyticus challenge. Our results show that V. alginolyticus infection resulted in dynamic changes of bacterial community composition in swimming crabs. Such changes were highlighted by the overwhelming overabundance of V ibrio and a significant fluctuation in the gut bacteria including the bacteria with high relative abundance and especially those with low relative abundance. These findings reveal that crab vibriosis gradually develops with the infection time of V. alginolyticus and tightly relates to the dysbiosis of gut bacterial community structure. This work contributes to our appreciation of the importance of the balance of gut bacterial community structure in maintaining the health of crustaceans.

关键词： Portunus trituberculatus vibrio alginolyticus gut bacterial community composition 16S rRNA gene amplicon sequencing

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Expression,Characterization and Immunogenicity of a Major Outer Membrane Protein from vibrio alginolyticus

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Acta Biochimica et Biophysica Sinica 2007年第3期39卷 194-200页

作者： Ronghua QIAN~(1,2) Wuying CHU~3 Zhijuan MAO~(1,4) Chongwen ZHANG~(1,2) Yongwei WEI~2 Lian YU~2* 1 Department of Biomedical Engineering,Zhejiang University,Hangzhou 310027,China 2 Institute of Preventive Veterinary Medicine,Zhejiang University,Hangzhou 310029,China 3 Institute of Subtropical Agriculture,Chinese Academy of Sciences,Changsha 410125,China 4 Ningbo Oceanography and Fishery Institute,Ningbo 315012,China Department of Biomedical Engineering Zhejiang UniversityHangzhou 310027 China Institute of Preventive Veterinary Medicine Zhejiang UniversityHangzhou 310029 China Institute of Subtropical Agriculture Chinese Academy of SciencesChangsha 410125 China Ningbo Oceanography and Fishery InstituteNingbo 315012 China

vibrio alginolyticus is one of the vibrio pathogens common to humans and marine *** infection and induction of the host immune response,outer membrane proteins of bacteria play animportant *** this study,an outer membrane protein gene(ompW)was cloned from *** andexpressed in Escherichia *** 645 bp open reading frame(ORF)encodes a protein of 214 amino acidresidues with a predicted molecular weight of 23.3 *** amino acid sequence showed a high identitywith that of Photobacterium damselae(96.2%)and vibrio parahaemolyticus(94.4%).The alignment analy-sis indicated that OmpW was highly *** dodecyl sulfate-polyacrylamide gel electrophoresisshowed that the gene was over-expressed in *** BL21(DE3).Western blot analysis revealed that theexpressed protein had *** recombinant protein was purified by affinity chromatographyon Ni-NTA Superflow *** yellow croaker vaccinated with the purified OmpW showed significantlyincreased antibody to OmpW,which could resist the infection by ***.A specific antibody wasdetected by enzyme-linked immunosorbent *** study suggested that the conserved OmpW could bean effective vaccine candidate against infection by ***.

关键词： vibrio alginolyticus ompW expression characterization immunogenicity vaccine candidate

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Starvation effects on pathogenic vibrio alginolyticus in natural seawater

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Acta Oceanologica Sinica 2008年第4期27卷 120-129页

作者： YI Jiabo CHEN Qiang ZOU Wenzheng YAN Qingpi ZHUANG Zhixia WANG Xiaoru Fisheries College Key Laboratory of Science and Technology for Aquaculture and Food Safety Jimei University Xiamen 361021 China First Institute of Oceanography State Oceanic Administration Qingdao 266061 China

To get a better understanding of the starvation survival strategy of pathogenic vibrio alginolyticus, log-phase cells were inoculated into sterile natural seawater for starvation studies. The results showed that all of total bacteria number, viable bacteria number and CFU number of V. alginolyticus increased remarkably at the initial starvation stage; after reaching their peaks at 5 d, both total bacteria number and viable bacteria number of V. alginolyticus fell slowly, while the CFU number fell more quickly after reaching its peak at 10 d; V. alginolyticus elongated their cells at the prophase of starvation, and then shrunk their volume and turned their shapes into ovals from rods at the anaphase of starvation; starved cells showed more sensitivity to heating and UV; starved cells showed no significant difference from unstarved ones at the lowest detection limit determined by indirect enzyme-linked immu- nosorbent assay （ELISA） ; starved cells＇ ability to adhere to the skin mucus of large yellow croaker （ Pseudosciaena crocea） showed a sharp decline as the starvation time increases; the cellular protein of V. alginolyticus increased remarkably at the ariaphase of starvation. The results indicated that pathogenic V. alginolyticus could survive in starvation for relatively long periods of time （ ≥2 months） in 28℃ natural seawater due to the morphological and physiological changes; however, starved V. alginolyticus cells showed less virulence and higher sensitivity under environmental stresses.

关键词： vibrio alginolyticus starvation survival adhesion ELISA resistance

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Immune response of Pseudosciaena crocea to the injection of vibrio alginolyticus

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Chinese Journal of Oceanology and Limnology 2009年第1期27卷 85-91页

作者：鄢庆枇张俊杰邹文政陈强庄峙厦王小如 Fisheries College of Jimei University Key Laboratory of Science and Technology for Aquaculture and Food Safety Fisheries College Ocean University of China First Institute of Oceanography State Oceanic Administration

For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension of vibrio alginolyticus in density of 2×107 CFU/ml, while each individual in the control group was injected i.p. with 0.2 ml sterile saline solution (0.85%). It was observed that the artificial injection of V. alginolyticus significantly increased the number of erythrocytes, leucocytes, lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of large yellow croaker, and significantly reduced the number of peripheral blood granulocytes as compared with those in the control group. No significant difference in acid phosphytase and superoxide dismutase activity of serum was detected between the two groups. It is suggested that non-specific immune factors including leucocytes and anti-bacteria substance in peripheral blood played important role at the initial stage of infection, and specific immune factors such as antibody then played important role in response to anti-infection at the latter stage.

关键词： Pseudosciaena crocea vibrio alginolyticus artificial infection immune response

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Adhesion of pathogenic vibrio alginolyticus to the gill mucus of Pseudosciaena crocea

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Acta Oceanologica Sinica 2007年第3期26卷 101-109页

作者： CHEN Qiang YAN Qingpi MA Shen ZHUANG Zhixia WANG Xiaoru Fisheries College Jimei University Xiamen 361021 China Fisheries College Ocean University of China Qingdao 266003 China First Institute of Oceanography State Oceanic Administration Qingdao 266061 China

Adhesion of vibrio alginolyticus to the gill mucus of Pseudosciaena crocea has been investigated using [ methyl-^3 H ] thymidine as isotope tracer. The results showed that： the adhesive quantity of V. alginolyticus increased with bacterial concentrations and reached equilibrium after incubated for 180 min; the higher adhesive quantity was obtained at 15 ~ 30 ℃ and sourish conditions; adhesion of V. alginolyticus could not achieved without Na^＋ , and Ca^2＋ played an auxiliary role in the bacterial adhesion; adhesion of V. alginolyticus was inhibited remarkably by starvation, heat treatment and periodic acid treatment; all of the eight kinds of carbohydrates investigated enhanced the adhesion of V. alginolyticus to the gill mucus of P. crocea, among them, glucose, mannose, fructose and maltose showed the specially enhanced adhesion. The results indicated that E alginolyticus could adhere to the gill mucus of P. crocea facilely in seawater, and this bacterial adhesion was influenced by environmental factors and closely related to superficial carbohydrate structures and some heat-sensitive structures.

关键词： vibrio alginolyticus adhesion Pseudosciaena crocea gill mucus

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Molecular characterization of an IL-1β gene from the large yellow croaker（Larimichthys crocea） and its effect on fish defense against vibrio alginolyticus infection

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Zoological Research 2015年第3期36卷 133-141页

作者： Jun WU Yu-Hong SHI Xue-Heng ZHANG Chang-Hong LI Ming-Yun LI Jiong CHEN Laboratory of Biochemistry and Molecular Biology School of Manne Sciences Ningbo University Ningbo 315211 China Donghai Sea Collaborative Innovation Center for Industrial Upgrading Mariculture Ningbo University Ningbo 315211 China

Interleukin 1β（IL-1β）, the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the c DNA and genomic DNA sequences of the IL-1β gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1β（Lc IL-1β） gene was most closely related to that of European seabass（Dicentrarchus labrax）, sharing 67.8% amino acid identity. In healthy large yellow croaker, Lc IL-1β transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon vibrio alginolyticus infection, Lc IL-1β transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant Lc IL-1β（r Lc IL-1β） improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, r Lc IL-1β induced monocytes/macrophages（MO/MΦ） chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that Lc IL-1β plays an important role in the large yellow croaker immune response against V. alginolyticus.

关键词： Interleukin 1β Large yellow croaker Survival rate vibrio alginolyticus Monocytes/macrophages

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Molecular Cloning and Bioinformatics Analysis of crp Gene in vibrio alginolyticus

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Agricultural Biotechnology 2022年第2期11卷 1-5,8页

作者： Zhiqing WEI Weijie ZHANG Linlin YIN Haiyun FENG Junlin WANG Fuyuan ZENG Xing XIAO Huanying PANG College of Fisheries Guangdong Ocean UniversityGuangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals/Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education InstitutionsZhanjiang 524088China

[Objectives]The crp gene in vibrio alginolyticus was studied.[Methods]The crp gene of *** HY9901 was cloned and analyzed by bioinformatics.[Results]The ORF of the crp gene is 633 bp long and the predicted amino acid sequence encompasses 210 amino acid *** physicochemical property analysis indicated that the chemical formula of CRP is C_(1051)H_(1704)N_(290)O_(308)S_(10) with a molecular weight of 23.6514 kDa,and its theoretical pI is ***,the protein is stable and *** protein had three protein kinase C phosphorylation site,three casein kinase II phosphorylation site,one N-terminal myristoylation *** BLAST analysis on the sequence revealed high homology with CRPs in other vibrio species,and particularly the sequence shares about a homology of 99.52%with the CRP in *** SWISS-MODEL software simulated the subunit tertiary structural model of the CRP,and the similarity with template 3hif.1.A was 95.71%.[Conclusions]This study provides a reference for the search for efficient protective antigens against vibrosis.

关键词： vibrio alginolyticus crp Bioinformatics analysis

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