拟态弧菌外膜蛋白OmpU 基因的原核表达及其免疫保护性研究

自行构建的pMD18T-OmpU重组克隆质粒经BamHI/EcoRI双酶切后,定向插入原核表达质粒pGEX-4T-1中构建重组表达质粒pGEx-4T-1-OmpU.将重组表达质粒转化至大肠杆菌E.coli BL21进行IPTG诱导表达.表达产物经SDS-PAGE电泳及Western-blot分析显示,以包涵体形式表达的GST-OmpU融合蛋白的分子量约为62.9 kD,可与鼠抗拟态弧菌外膜蛋白(Omps)免疫血清发生特异性反应,提示重组OmpU保留了天然Omps的抗原性.用纯化的融合蛋白免疫昆明系小鼠,以50LD50拟态弧菌(5×103CFU/mL)攻击,小鼠的免疫保护率为60%(9/15).表明OmpU是拟态.弧菌的保护性抗原之一,具有研发成为外膜蛋白基因工程亚单位苗的潜在价值.     

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.     

Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus (Bloch)

A 750-bp internal fragment of the alkaline serine protease gene (asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5'- and 3'- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD(50) of 0.25 microg protein g(-1) body weight.     

哈氏弧菌外膜蛋白与溶藻弧菌脂多糖偶联疫苗的效果研究

采用十二烷基肌氨酸钠法提取哈氏弧菌(Vibrio harveyi)SpGY020601株的外膜蛋白(OMPC),采用酚水法提取溶藻弧菌(Vibrio alginolyticus)EpGS021001株的脂多糖(LPS).通过碳化二亚铵(EDC)介导的缩合反应,将哈氏弧菌的OMPC与溶藻弧菌的LPS偶联.OMPC-LPS偶联物、未偶联的哈氏弧菌OMPC、溶藻弧菌LPS、哈氏弧菌OMPC和溶藻弧菌LPS的简单混合物以及生理盐水按相同程序免疫卵形鲳鲹(Trachinotus cvatus).检测血清溶菌酶活性、血清抗体微量凝集反应结果显示,卵型鲳鲹经OMPC-LPS偶联物、OMPC、LPS以及二者的简单混合物免疫后,各免疫组间血清溶菌酶活性没有显著差异(P＞0.05),但均显著高于生理盐水对照组(P＜0.05);OMPC-LPS偶联物免疫组的血清抗溶藻弧菌EpGS021001抗体效价较单纯溶藻弧菌LPS免疫组、简单混合物免疫组出现得早,抗体滴度高;与此类似,OMPC-LPS偶联组中抗哈氏弧菌SpGY020601的血清抗体效价较单纯哈氏弧菌OMPC组、简单混合组出现得早,抗体滴度高,且持续时间长;对EpGS021001、SpGY020601的攻击,OMPC-LPS偶联物免疫组的保护率分别为85%和95%,高于单纯溶藻弧菌LPS免疫组的70%、单纯哈氏弧菌OMPC免疫组的75%,也高于简单混合物免疫组的80%和82.5%.     

Development of novel aptamer‐based enzyme‐linked apta‐sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 10⁴/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.     

Enhanced immune responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in orange‐spotted grouper (Epinephelus coioides)

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK‐OmpU fusion protein in addition to the metabolizable Montanide^TM ISA 763 A VG adjuvant were developed and evaluated in the orange‐spotted grouper. The results indicate that recombinant V. harveyi protein‐based vaccines resulted in a remarkably higher expression of IL‐1β and IL‐8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine‐injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK‐OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK‐OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.     

Effects of crude extracellular protein and crude intracellular polysaccharides of Vibrio alginolyticus on the growth,energy metabolism regulation and WSSV resistance of Litopenaeus vannamei

This study aimed to investigate the effects of adding crude extracts of the extracellular protein (Ex‐Pro) and intracellular polysaccharides (In‐Poly) of Vibrio alginolyticus to diets, at a dosage of 10 g/kg, on the growth, energy metabolism and WSSV (White Spot Syndrome Virus) resistance of Litopenaeus vannamei (initial weight, 0.88 ± 0.04 g/shrimp). Growth and survival rate were not significantly affected by the crude extracts (p > 0.05). Shrimp fed Ex‐Pro crude extracts showed higher succinate dehydrogenase (SDH) activity in the hepatopancreas and muscle but lower lactate dehydrogenase (LDH) activity in the muscle (p < 0.05). The activities of hexokinase (HK), pyruvate kinase (PK), LDH and SDH in the hepatopancreas and the activity of SDH in the muscle were significantly increased by feeding In‐Poly crude extracts (p < 0.05). In contrast, the content of fatty acid synthase (FAS) in the muscle was significantly reduced by the crude extracts (p < 0.05). The contents of glucose and triglyceride and the activity of the electron transport system in the hepatopancreas were significantly increased by the crude extracts (p < 0.05), and the WSSV resistance of the shrimp was increased. These results indicated that the Ex‐Pro and In‐Poly crude extracts of V. alginolyticus could affect energy metabolism, and there was a correlation between WSSV resistance and energy metabolism in L. vannamei.     

副溶血弧菌ZJ2003株两种铁调外膜蛋白的克隆、表达和免疫原性

从浙江省象山港网箱养殖大黄鱼病鱼分离的一株副溶血弧菌(Vibrio parahaemolyticus)ZJ2003中克隆了两种铁调外膜蛋白psuA和pvuA的基因(GenBank登录号:DQ141607、DQ141608),经PCR的方法去除两基因的信号肽编码序列后亚克隆到原核表达载体pET30a( )中,经IPTG诱导获得大量表达。表达的重组蛋白以包涵体形式存在。包涵体蛋白经尿素法纯化及梯度透析法复性后以100μg/尾的剂量免疫大黄鱼,4周后经活菌攻毒获得80%的免疫保护率。免疫印迹分析表明,人工感染存活鱼的血清可以识别此两种重组蛋白。研究结果显示该两种铁调外膜蛋白具有良好的免疫原性,有可能作为高效疫苗成份。     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

In vitro protein digestibility of different grow‐out stages of spotted rose snapper (Lutjanus guttatus,Steindachner, 1869)

The aim of this study was to compare in vitro protein digestibility between two groups of fish, at early (21 g) and late stages (400 g) of spotted rose snapper Lutjanus guttatus, to evaluate the degree of hydrolysis (DH) and total amino acid release (TAAR) using crude extracts from stomach, pyloric caeca and intestine of 13 protein ingredients including marine, animal and plant meals. Degree of hydrolysis and TAAR were measured by a pH‐Stat method, and the PAGE‐Zymogram was also used as complementary technique. Differences in DH were found between both grow‐out stages mainly in the alkaline hydrolysis phase. Fish and squid meals (marine sources) had the highest DH and TAAR, followed by porcine meat and poultry meal by‐products from recycling sources, and soybean and canola meals (plant sources), which represent better protein sources for use in practical diets. Stomach zymograms showed two pepsin isoforms in both grow‐out stages. Pyloric caeca and intestine zymograms showed five bands with proteolytic activity in the early grow‐out stage, whereas four additional bands were found in late grow‐out stage. Alkaline proteases were identified as serine and metalloproteases. Thus, L. guttatus presents an ontogenetically differentiated digestive enzyme pattern that modifies the DH and TAAR of different protein sources.     

哈维氏弧菌外膜蛋白OmpW基因克隆、表达及DNA疫苗的免疫效果

参照GeneBank上登录的哈维氏弧菌外膜蛋白OmpW基因序列,设计引物扩增OmpW的开放阅读框,序列分析结果显示该基因全长645 bp,编码214个氨基酸。将其定向克隆到原核表达载体pET-32a( ),在大肠杆菌BL21中成功表达出带His-tag的融合蛋白,融合蛋白分子量约为43.8 ku,且主要以包涵体形式存在。优化的表达条件为温度37 ℃,IPTG浓度0.1 mmol/L,诱导时间5 h。将纯化的融合蛋白免疫SPF昆明小鼠制备多克隆抗体,ELISA方法检测抗体效价达1:30,000,Western-blot结果表明鼠抗OmpW血清能与诱导后的重组蛋白发生特异反应,提示OmpW可能是哈维氏弧菌的一种重要保护性抗原。为了进一步研究哈维氏弧菌外膜蛋白OmpW基因DNA疫苗对红笛鲷的免疫保护效果,构建了真核表达重组质粒pCDNA-OmpW,然后将真核质粒免疫接种红笛鲷。PCR结果显示,免疫接种第7、28 d,在红笛鲷的肌肉、头肾、肝脏和脾脏组织均存在质粒分布;RT-PCR结果显示,免疫接种第7、28 d,红笛鲷不同组织均有目的基因的表达。ELISA结果表明,红笛鲷体内血清产生了抗OmpW蛋白的高效价抗体。Western- blot分析表明,DNA疫苗免疫后红笛鲷体内表达了相应的目的蛋白,并诱导产生了相应抗体。攻毒试验结果表明,免疫保护率高达60 %,可见pCDNA-OmpW可作为红笛鲷预防哈氏弧菌感染的有效候选疫苗之一。     

Effect of incubation temperature on the embryonic development and yolk‐sac larvae of the Pacific red snapper Lutjanus peru (Nichols & Murphy, 1922)

The effect of incubation temperature on embryonic development and yolk‐sac larva of the Pacific red snapper Lutjanus peru were evaluated by testing the effect of 26, 28 and 30°C, as this is the natural thermal interval reported during the spawning season of Pacific red snapper in the Gulf of California, Mexico. Sixteen developmental stages were observed. The incubation temperature affected the rate of development and time to hatching, being shorter at 30 than at 26°C, but no significant effect (P < 0.05) on larval length at hatching was registered. The depletion rate of yolk sac and oil globule was affected by incubation temperature particularly during the first 12 h post hatching (hph). At the end of the experiment (48 hph), significantly (P < 0.05) larger larvae were recorded at 26°C (TL = 3.22 ± 0.01 mm) than at 28° (TL = 3.01 ± 0.02 mm) and 30°C (TL = 2.97 ± 0.05 mm). Incubation of newly fertilized eggs at 26°C produces larger larvae, which may help to improve feeding efficiency and survival during first feeding.     

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.     

溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护研究

为研究溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护作用,实验构建了重组真核表达质粒pcDNA-flaC并将该质粒肌肉注射红笛鲷,采用PCR、RT-PCR、ELISA和攻毒试验等方法检测了该真核表达质粒在红笛鲷组织内的分布、表达和对红笛鲷的免疫保护.PCR结果显示,免疫接种7和28 d,注射点周围肌肉、鳃、肾脏、肝脏和脾脏都存在质粒分布;RT-PCR结果显示,免疫接种后第7天、14天和28天,红笛鲷不同组织内均有目的基因表达.ELISA结果表明,鱼血清内产生了抗FlaC蛋白的抗体,表明DNA疫苗免疫后鱼体表达了目的蛋白,并诱导产生了相应抗体.攻毒实验表明,免疫后的红笛鲷能较好地抵抗致病性溶藻弧菌的感染.结果表明,质粒pcDNA-flaC可能是抵抗溶藻弧菌感染的有效的疫苗候选物.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

Development of a two‐step,non‐probed multiplex real‐time PCR for surveilling Vibrio anguillarum in seawater

Vibrio anguillarum is an aggressive and halophilic bacterial pathogen most commonly originating from seawater. Vibrio anguillarum presence in fisheries and aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from haemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. This study served to develop a non‐probe, multiplex real‐time PCR assay to rapidly detect V. anguillarum presence in seawater. Specific primers targeting genes vah1, empA and rpoN of V. anguillarum were selected for multiplex reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time as well as annealing time and temperature of DNA amplification were optimized, thus reducing reaction duration. The two‐step, non‐probed multiplex real‐time PCR set forth by this study detects as little as 3 CFU mL^?1 of V. anguillarum presence in sea water, without enrichment cultivation, in 70 min with molecular precision and includes melting curve confirmation.