重组LTB蛋白在水弧菌VSP60中的高效表达与纯化

目的 优化重组大肠杆菌热不稳定肠毒素B亚单位 (rLTB)工程菌 (VSP6 0 )高效表达的条件。方法 以UVP凝胶照相系统扫描以及改良Lowry′s法检测rLTB表达量并分析各种因素对其影响。利用SephacrylS 10 0凝胶层析纯化rLTB蛋白。结果 工程菌 30℃振荡培养至吸光度 (A6 0 0 )值达0 .2～ 0 .3时加IPTG(终浓度为 0 .5mmol L) ,诱导培养 18～ 2 2h为rLTB蛋白表达的最佳条件。SephacrylS 10 0凝胶柱一步层析后 ,rLTB蛋白纯度可达 98.1%。结论 本研究所用的培养和纯化工艺简单易行 ,可获得高产量、高纯度的rLTB蛋白。

Vaccination in three different ways against vibriosis of Seriola dumerili caused by Vibrio hollisae

Bacterin was prepared by formalin-inactivating the virulent strain of Vibrio hollisae isolated from diseased Seriola dumerili （amberjack） suffering from vibriosis. Healthy S. dumeriIi were vaccinated by respective procedures of intramuscular injection, immersion, and orally administration. Results of the three different vaccinations were compared. Blood was drawn from the vaccinated fish every 7 days, and the antibody titers and lysozyme activities of the sera were determined. The antibody titer of injected fish was 1：40 at 7 d, and reached its peak of 1：320 at 28 d, while the fish vaccinated by immersion and orally administration exhibited weak antibody responses, the antibody titres of 〈1：10, 1：20, 1：160 were observed at 7 d, 14 d, 35 d respectively. Compared with the control, the vaccinated fish exhibited significantly higher lysozyme activities （P〈0.05）. Upon challenge with virulent strain, the relative percent survival （RPS） of injected, immersed and oral administrated fish were 75%, 45%, and 40% respectively, and the injected fish showed significantly higher RPS than immersed and oral administrated fish. The results suggested that vaccination of S. dumerili by the injection would be the best strategy to prevent the vibriosis in S. dumerili farm.

Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene （rDNA） fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.

Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coil

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, hetemlogous expression of the empA gene encoding metallopmtease and export of the recombinant metallopmtease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide （611 amino acids） consisting of four domains： a signal peptide, an Nterminal pmpoptide, a mature region and a C-terminal pmpoptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metallopmtease as the mature pmtease was further confirmed by SDS-PAGE and im- munoblotting. It was found that recombinant metallopmtease with the EmpA activity and antigenicity was exported into the poriplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.

Phenotypic and Genotypic Comparison of Vibrio in Seawater Fish from Batam and Mataram, Indonesia

Grouper and snapper are the potential fishery commodity in Indonesia with a high economic value, as well as an export commodity. A common disease in grouper and snapper aquaculture is vibriosis. Vibriosis is a disease caused by bacteria of the genus Vibrio. The aim of study was to compare between phenotypic and genotypic identification of Vibrio isolated from Batam and Mataram, Indonesia. Bacteria were isolated from anterior kidney and eye of fish, then grown in thiosulfate-citrate-bile salts-sucrose （TCBS） and incubated in room temperature （25-28 ~C） for 24 h, and identified using morphology and biochemical test. Bacterial isolates were extracted, amplified and sequenced on 16S rRNA region. Phylogenetic tree of bacteria was constructed using neighbor-joining and maximum-parsimony methods. The phenotypic identification was found six isolates of Vibrio from Batam, such as K alginolyticus, V. carchariae, K damselae, V. fluvialis, V. furnissii and K parahaemolyticus. Three isolates were found from Mataram, such as 1I. alginolyticus, V. carchariae and V. fluvialis. Blast analysis showed isolates of V. alginolyticus_btm and V. carchariae_btm homolog to V. parahaemolyticus strain DAHMV3; isolates of V. damselae_btm and K alginolyticus_mtr homolog to V. neocaledonicus strain MS1; isolates of V. parahaemolyticus__btm and V. furnisii_btm homolog with Photobacterium damselae subsp, damselae strain： 04Ya311 and isolate of K fluvialis_mtr homolog to V. azureus strain MMRF532, respectively. All phenotypic identification was not supported by molecular identification on 16S rRNA region. It was suggested that phenotypic identification should be supported by molecular examination, especially in identification of Vibrio species.