水生动物病害数字化标本库的建设及其在水生动物医学专业上的应用探讨

水生动物病害标本是水生动物医学专业实验课的重要实验材料,是加深学生对水生动物疾病的临床表现、致病机制认知的重要教学资源。为了有效地解决水生动物病害标本库建设中标本数量不足、标本制作不规范、病害标本不典型、病原鉴定不明确、标本保存不当等问题以及标本在实验教学中展示模式单一、学习途径有限等问题,从标本库现有问题的分析、实体标本库的改造、数字化标本的建造、数字化标本在教学中的应用4个方面介绍了水生动物病害数字化标本库的建设以及标本在水生动物医学专业上的应用。实践表明,借助数字化标本库的应用,教师讲课更加生动形象,学生学习兴趣更浓更方便,教学质量得到了显著提高。

Construction of Live Attenuated Vaccine against Vib- rio alginolyticus and the Evaluation on Its Immuno- protection Effect

Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system （T3SS）, an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface, vscO locates at the ＂needle＂ site of T3SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mu- tant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol （Cm） and sucrose screening. The LDs0 changes of ZJO3AvscO mutant strains compared with the strain ZJ03 were e^amined in grouper （ Epinephelus coioides）. The ZJO3AvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJO3AvscO in E. coioides through injection and immersion, the spe- cific antibody titers were markedly higher than that in the saline control group （P 〈 0.05 ）. The titers of injection and immersion group on the forth week reached the maximums at 1：2 048 and 1： 128, respectively. The relative percentage survival （RPS） of injection group was 84%, while that in immersion group was 68%. These results indicate that the ZJO3AvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus.

Molecular Cloning and Bioinformatics Analysis of kdpE Gene from Vibrio alginolyticus

The two-component regulatory system plays an important role in the growth and virulence of bacteria. In this study, the phosphate transcriptional regulatory protein （KdpE） gene of Vibrio alginolyticus strain HY9901 was cloned. Sequence analysis revealed that the length ofkdpE gene is 732 bp and encodes a putative protein of 243 amino acids. The predicted molecular weight （MW） of KdpE was 27.66 kD with an estimated pI of 5.01. Using SignalP 4.0 and TM- HMM Server 2.0 software, it was predicted that the KdpE protein was located in cytoplasm. It did not contain a signal peptide or a transmembranous region. A phylogenetic tree was constructed by MEGA 5.0 software, indicating that the KdpE of V. alginolyticus bad high genetic relationship with Vibrio campbellii and Vibrio parahaemolyticus. Using SWISS-MODEL work-space, the three-dimensional structures of conserved domain REC in the KdpE was determined. These results can provide a basis for further studies on the two-component regulatory systems of V. alginolyticus.

湛江卵形鲳鲹致病链球菌的分离、鉴定及药敏实验

为了确定2017年6月至2017年9月湛江市太平镇网箱养殖卵形鲳鲹(Trachinotus ovatus)疑似发生鱼链球菌病的病原菌及其毒力特性,取病鱼的脑、肝脏、脾脏和肾脏在血平板上进行分离培养,将分离培养的病原菌对卵形鲳鲹进行人工回归感染实验,试验鱼呈现自然发病症状:游泳异常、眼球突出且浑浊、鳍条基部充血,证明分离菌为卵形鲳鲹爆发性流行病的致病菌。通过形态学观察、生化鉴定、16S rRNA序列分析,结果表明,分离的致病菌为海豚链球菌(Streptococcus iniae),暂定为中国湛江株GHZ007(GenBank:KX788919),其对卵形鲳鲹的LD50为2.42×106CFU/mL。药敏实验结果表明,分离菌株对四环素类和氨基糖苷类药物等17种试验药物敏感,对青霉素、复方新诺明等8种试验药物有耐药性。本研究将有助于为海豚链球菌病的防治提供理论基础。

斜阳岛珊瑚礁区海泥可培养细菌的分离鉴定及其ECP抑菌作用

为探究斜阳岛珊瑚礁区海洋微生物的代谢产物活性,为天然材料制备新药物资源的开发提供一定的理论依据和实践指导,本研究从斜阳岛珊瑚礁区海泥分离纯化得到3株菌株,经鉴定分别为蜡状芽孢杆菌(Bacillus cereus)、Sphingopyxis terrae和Fictibacillus phosphorivorans,分别培养并提取3株细菌的胞外产物(extracellular products,ECP),与硝酸银制备成生物纳米银,OD法测定其抑菌(溶藻弧菌)动力曲线,用2倍稀释法测出3株菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)分别是21.2μg/mL、18.8μg/mL、20.0μg/mL,用牛津杯法分别测定了3株菌与溶藻弧菌的抑菌活性,抑菌直径分别是(12.20±0.18)mm、(1.32±0.12)mm、(10.81±0.15)mm,安全性实验表明此3株菌对鱼体是安全的。本实验对斜阳岛珊瑚礁区海泥中可培养细菌进行了初步分离与鉴定,并利用分离菌的胞外产物合成生物纳米银,结果显示其具有一定的抑菌效果,从而证明其具有开发新的天然防腐剂和新药的潜力。

溶藻弧菌Ⅲ型分泌系统效应蛋白HY322基因的克隆和生物信息学分析

本研究从溶藻弧菌中成功克隆获得HY322基因的编码区序列（GenBank：KX245317.1）,该基因全长969 bp,可编码322个氨基酸,应用生物信息学的方法和工具对溶藻弧菌HY9901效应蛋白HY322的理化性质、蛋白结构、遗传进化关系和抗原特性等方面进行预测和分析。结果表明：HY322蛋白为不稳定的亲水性蛋白,蛋白呈酸性,无跨膜区,无明显的信号肽;二级结构以α螺旋为主,进化分析显示溶藻弧菌HY9901与哈维氏弧菌（V.harveyi）聚为一支,表明在进化关系上最为接近。HY322序列中包含有一个与鞭毛形成有关的Fli N功能结构域。多种参数预测了HY322可能的B细胞抗原优势表位,分别是第32~33、100~102、138~140、215~216、235~238、246~249区段。利用SWISS-MODEL软件,得到了HY322亚基三维模型。本研究从生物信息学角度验证了HY322作为弧菌共同抗原的可行性,为下一步的疫苗研发提供了理论依据。