鱼生长激素基因在大肠杆菌中表达水平的研究

采用聚合酶链反应（ＰＣＲ）技术对鲤和鲑生长激素ｃＤＮＡ的５‘端和３’端进行定向改造。将改造后的５种基因分别克隆到大肠杆菌表达质粒ｐＢＶ２２０进行原核表达,以研究鱼生长激素基因在大肠杆菌中的表达水平。实验结果表明：（１）核糖核蛋白体结合位点（ＳＤ）与起始密码子ＡＵＧ之间的距离对鱼生长激素的表达水平有影响;（２）鲑鱼生长激素基因的终止密码子ＵＡＧ可能不会有效终止该基因在大肠杆菌中翻译的进行而造成部分通     

Survival of the fish pathogen Aeromonas salmonicida in the marine environment

Abstract. The fate of the fish pathogen Aeromonas salmonicida in the marine environment was examined by culture, epifluorcscence microscopy, the indirect fluorescent antibody technique (1FAT), direct viable counts (= Kogure technique) and the assessment of respiratory activity by the reduction of tetrazolium compounds to coloured formazans. In laboratory- based systems containing sediment, water, algae and invertebrates, cells of A. salmonicida were observed microscopically after plate counts on tryptone soya agar declined to zero. Survival was maximal at a salinity of 25%_o, and in wood and sediment, notably mud, rather than the water column.     

High prevalence of antimicrobial resistance in Escherichia coli,Salmonella spp. and Staphylococcus aureus isolated from fish samples in India

Antibiotic resistance, driven by inappropriate use of antimicrobial agents for fish farming, is an expanding global health threat. Demand of animal protein for human consumption is entraining at remarkable pace which in turn, upturning the non‐judicial use of life‐saving drugs in these modern animal production practices at an incautious rate. Hence, study was devised to observe the antimicrobial resistance (AMR) pattern of human prominence bacteria, against drugs of critically and highly importance of both human and veterinary medicine, circulating over the fish farming in India. One hundred sixty fish faecal samples were obtained from distinct geographical locales of Rajasthan, India. Overall, 202 isolates comprising of Escherichia coli (81, 51%), Salmonella spp. (72, 45%) and Staphylococcus aureus (49, 31%) were isolated to screen for resistance. Antimicrobial resistance has been analysed using broth microdilution method. In case of E. coli and Salmonella spp., highest resistance (>95%) was observed against streptomycin while for S. aureus, it was observed against trimethoprim, along with high resistance to other antibiotics. Majority of the isolates were found to be resistant to more than three antibiotics implying multi‐drug resistance (>89%) relative to the critical and highly important antibiotics. The high AMR is a reflection of misuse of these agents in the fish farming, which is a concern of serious health issues. Our findings persuade the prudent use of antimicrobial agents in fish farming and other aquaculture settings that may assist in drafting of the new policies for the judicial use of these life‐saving drugs in these sectors.     

牦牛大肠杆菌病的研究进展

大肠杆菌是严重危害牦牛养殖业的重要疾病,由于其血清型复杂和抗生素的大量使用所导致耐药菌株不断增加,对肠道正常菌群造成不良影响,给防制工作带来了巨大的困难和影响。据统计,该病在西藏牦牛中的发病率为10.1%,死亡率为2.5%,致死率为45.0%。西藏农牧学院动物科学学院曾群辉课题组对牦牛大肠杆菌的生物学特性进行了深入研究。所研制出的牦牛大肠埃希氏菌多价油佐剂甲醛灭活疫苗具有良好的免疫原性,对预防该病奠定了基础。     

The ability of carnobacteria isolated from fish intestine to inhibit growth of fish pathogenic bacteria: a screening study

In the present study a microtitre plate assay was used to evaluate antagonistic activity of 157 intestinal bacteria belonging to Carnobacterium isolated from Arctic charr (Salvelinus alpinus L.), Atlantic salmon (Salmo salar L.) and wolf fish (Anarhichas lupus L.) against fish pathogenic bacteria. One hundred and forty‐nine strains isolated from Arctic charr fed; (a) different lipid levels and (b) different fatty acids were screened for their ability to inhibit growth of the fish pathogen Aeromonas salmonicida ssp. salmonicida strain AL 2020 (the causative agent of furunculosis). Carnobacterium maltaromaticum and Carnobacterium mobile isolated from fish fed a low‐lipid diet inhibited growth of the pathogen, while none of the Carnobacterium divergens isolated from fish fed the high‐lipid diet had this ability. When Arctic charr was fed different fatty acids, was the frequency of antibacterial ability of C. maltaromaticum highest in strains isolated from fish fed 4%α‐linolenic acid (18:3 n‐3) and lowest in strains isolated from fish fed 4% linoleic acid (18:2 n‐6). Extracellular growth inhibitory compounds harvested in exponential and stationary growth phase from eight carnobacteria strains isolated from three fish species were tested for their ability to inhibit growth of six fish pathogens [A. salmonicida, Vibrio splendidus strain VS11, Vibrio salmonicida strain LFI 315, Vibrio anguillarum strain LFI 317, Moritella (Vibrio) viscosa strain LFI 5000 and C. maltaromaticum (piscicola) CCUG 34645]. The highest antibacterial activity was found when cellular extracts of the producer isolate were harvested in stationary growth phase. Scanning electron microscopy (SEM) investigations of A. salmonicida showed that cell morphology was affected by the inhibitory substance produce by strain 8M851, a Carnobacterium inhibens‐like bacteria.     

Nutritional regulation of intestine morphology in larval cyprinid fish, silver bream (Vimba vimba)

The present study includes the evaluation of morphological changes in the digestive tract of larval, stomachless fish silver bream ( Vimba vimba ) fed with various diets – live Artemia nauplii, commercial feed Aglo Norse (NOR) and semi-purified formulated diets: casein–gelatin (CG), dipeptide-protein (50P), dipeptide (100P), no-arginine dipeptide diet (100Pw/oArg) and a free amino acid (FAA) mixture diet. The supranuclear area of enterocytes in the posterior intestine contained enlarged absorptive vacuoles in the FAA, 100P and 100Pw/oArg groups, compared with the remaining groups. Hepatocytes' cytoplasm in fish fed with FAA, 100P and 100Pw/oArg contained mainly glycogen, and no lipid vacuoles were found. Fish fed with 100Pw/oArg showed the lowest hepatocyte surface areas while in those fed with 50P, the largest nuclei diameters were observed. Fish fed with Artemia , NOR and CG diets showed significantly ( P <0.05) higher number of proliferating cells compared with the remaining groups. Chromogranin A staining showed endocrine-immunoreactive cells (CgA-IR) in the taste buds in the oral cavity and in the enterocytes' supranuclear areas of the anterior and posterior intestine. We conclude that the growth rate and histological examination of the digestive tract in the 50P group of silver bream showed no nutritional deficiency.     

鸡大肠杆菌病病原的分离与鉴定

无菌采取病死雏鸡肝、脾、肺等脏器病料,进行了大肠杆菌病原的分离培养与鉴定。综合流行病学、临床症状、病理变化和实验室诊断,确诊为鸡大肠杆菌病。同时,通过药敏试验筛选出敏感药物,为以后的治疗与预防提供理论依据和实际应用参数。     

鱼类肠道组织结构、功能、影响因素及其保护物质的研究进展

肠道可以吸收营养物质并抵御病原体、微生物和细菌毒素进入内部环境,因此在鱼类健康中起着重要作用。作为鱼类与外部环境接触面积最大的器官,肠道受到多种因素的侵害,严重制约了鱼类的健康生长。文章从鱼类肠道组织结构、肠道的屏障功能、引起肠道损伤的因素以及有效缓解肠道损伤的物质等四个方面进行综述,可为进一步了解鱼类肠道健康的分子机制及相关产品的研发提供参考。     

pH对乌鳢胃、肠、肝胰脏蛋白酶活性的影响

设定胃蛋白酶活性在pH2.2~4.6、肠及肝胰脏蛋白酶活性在pH6.4~8.8范围内测定。pH各5个梯度,梯幅0.6。乌鳢胃、肠、肝胰脏蛋白酶的活性最适pH值为别为2.8、8.2、8.2。在最适pH值时酶活性的大小顺序为胃>肠>肝胰脏。pH对胃蛋白酶的影响大于对肠和肝胰脏蛋白酶的影响。     

补体和溶菌酶在清除文昌鱼体内细菌中的作用

文昌鱼作为研究脊椎动物起源和进化的模式生物,由于缺乏自由循环的血细胞,关于其清除体内病原体的机制,迄今尚无报道。采用体内细菌清除试验、体外杀菌试验、特异性抗体抑制试验、抑制补体途径试验及Western blot方法,研究了文昌鱼体内细菌清除机制及动力学。试验结果表明,文昌鱼能有效清除入侵的异源微生物,体液对大肠杆菌具有较强的杀灭活性。补体和溶菌酶是参与清除入侵大肠杆菌的主要因子,通过替代途径激活的补体较溶菌酶作用更强,这是关于文昌鱼清除体内细菌机制及其相关因子的首次报道。     

冷冻时间对贝类大肠杆菌、细菌总数结果的影响

研究探讨了贝类以缢蛏为代表在室温16℃、湿度50%～70%条件下,不同冷冻时间对贝类大肠杆菌、细菌总数的影响。结果表明,随着存放时间延长,贝类菌落总数呈下降趋势,变化范围分别在1.0×102～1.0×103 CFU/g之间。而大肠杆菌数基本小于30MPN/100g,低于农业部无公害贝类标准。存储8d以后,大肠杆菌数量无明显变化,而细菌总数在6d以后呈下降趋势。可见贝类在-11～-18℃的温度下,大肠杆菌数存储8d无明显变化,细菌总数存储6d无明显变化。     

鸡抗鸭源大肠杆菌卵黄抗体的研制

用鸭源大肠杆菌125号菌株制成的灭活油佐剂苗、蜂胶苗及无佐剂菌苗作为免疫原,免疫经产蛋鸡,定期收集鸡蛋,用氯仿提取卵黄抗体,采用试管凝集法检测抗体效价。结果表明:油佐剂灭活苗的抗体维持时间较长,在免疫后60d还能保持较高水平,其峰值达到1∶24。蜂胶灭活疫苗的抗体产生速度比较快,消失也较快,其峰值达到1∶12,40d后抗体基本上消失。不加佐剂的灭活菌苗抗体产生速度较快,10d达到峰值1∶16,然后呈逐渐下降趋势,免疫40d后波动较小。结果,油佐剂灭活菌苗免疫后产生的抗体效价及持续时间高于无佐剂灭活苗和蜂胶苗的抗体效价。     

泰州地区禽源性大肠杆菌的分离与鉴定

为摸清泰州地区禽大肠杆菌的流行情况及其耐药性,我们于2005年初至4月间,从泰州地区的鸡场、鸭场具有典型大肠杆菌病病变的病死鸡、鸭体内分离到14株大肠杆菌,进行药物敏感性试验,结果显示14个菌株对壮观霉素、阿米卡星高度敏感。血清型鉴定结果显示其O血清型包括O 1、O 2、O 78、O 107。这些菌株均为高致病力毒株。     

1个白斑综合症病毒基因的克隆及其在大肠杆菌M15中的表达

根据对虾白斑综合症病毒（white spot syndrome virus，WSSV）1个可能编码蛋白的开放读码框（open reading frame，ORF）的序列（WSSV A基因），结合pQE30载体的多克隆位点，设计1对引物进行PCR，扩增出大小为0.28kb的A基因片段。把目的片段克隆到pGEM-T Easy载体上，构建出重组质粒pGT-A，再用引物两端酶酶切出目的片段，并按正确的读码框顺序插入到pQE30表达载体上的乳糖操纵子中，构建出带有目的片段的重组质粒pQE30-A，然后将重组质粒转化到大肠杆菌M15细胞，经IPTG诱导，SDS-PAGE和Western blot显示有与A基因预期大小11kD相吻合的融合蛋白带。结果表明，来源于WSSV的这一ORF是1个可表达的基因。     

斜带石斑鱼生长激素cDNA克隆及其在大肠杆菌中的融合表达

通过构建斜带石斑鱼垂体cDNA文库,克隆了其生长激素(GH)全长cDNA。斜带石斑鱼GH全长cDNA为955bp,编码的多肽为204aa。应用PCR方法把编码GH成熟肽的cDNA片段克隆到表达载体pET-15b,在大肠杆菌BL21(DE3)表达N端含6个组氨酸的融合多肽。SDS-PAGE结果表明,0.4mmol·L-1IPTG诱导表达的蛋白约为24kDa,主要为不溶性的包含体。细菌裂解液沉淀溶于6mol·L-1盐酸胍后,用Ni2+-NTA树脂进行亲和分离纯化,纯化产物在SDS-PAGE上表现为一条24kDa的蛋白带。在黑鲷GH放射免疫分析系统中,纯化产物能与黑鲷GH竞争结合GH抗体,表明大肠杆菌表达的斜带石斑鱼GH融合多肽具有GH免疫活性。     

Expression of recombinant triangular bream (Megalobrama terminalis) pre-IGF-I in Escherichia coli

The insulin-like growth factor I (IGF-I) cDNA (GenBank Accession No. AY247412) of triangular bream (Megalobrama terminalis) was expressed for the first time in Escherichia coli. To construct the expression plasmid, the IGF-I cDNA was subcloned into prokaryotic-expressing vector pGEX-4T-1. The E.coli JM109 was transformed with the recombinant plasmid pGEX-4T-1-IGF-I, and the transgene expression was observed after being induced with isopropyl-β-D-thiogalactoside (IPTG). The results of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting indicated that the recombinant fusion protein had immune activity and the molecular weight was about 47 kDa. The results of SDS-PAGE and thin layer scanning showed that the yield of fusion protein had been enlarged with prolonging time. When the time of induced expression was 1, 2, 3, 4, 5, and 6 h, the expression amount was approximately 1.4, 4.3, 8.1, 11.3, 16.3 and 18.8% of total bacterial protein respectively.     

Survival,persistence and proliferation of Vibrio anguillarum in juvenile turbot,Scophthalmus maximus (L.), intestine and faeces

The aim of this study was to determine whether orally administered V. anguillarum cells could survive passage through the intestinal tract of feeding turbot, and thereafter, proliferate in the released faeces. In addition, the growth of the pathogen in turbot faeces in the presence of a Carnobacterium sp. with inhibitory effects against a number of bacterial fish pathogens was studied. It was found that V. anguillarum cells survived the acidic environment of the stomach for several hours, persisted in the intestine and readily proliferated in the released faeces. The antagonistic Carnobacterium sp. inhibited the growth of V. anguillarum during co-culture in turbot faecal extracts. From the results, it was concluded that the turbot intestinal tract and faeces can serve as an enrichment site for V. anguillarum, and the use of intestinal bacteria with antagonistic activity against vibrios may be used to reduce the load of fish pathogenic vibrios in turbot hatcheries.     

Is the turbot,Scophthalmus maximus (L.), intestine a portal of entry for the fish pathogen Vibrio anguillarum?

The role of the intestinal tract in Vibrio anguillarum infection of turbot, Scophthalmus maximus (L.), fingerlings was investigated in two in vivo models and the possible mechanisms involved were studied in vitro. Viable V. anguillarum cells were detected in spleens from more than 50% of the fish administered the pathogen orally or rectally, suggesting that the intestinal tract is a portal of entry for V anguillarum. In transmission electron micrographs, V. anguillarum- like cells were seen close to the rectal epithelium, suggesting penetration of the mucus layer, but no epithelial cell penetration or endocytosis was evident. Attachment to intact turbot intestines was investigated, and 80% or more of the bacterial cells still remained attached after serial washings. A significantly higher number of cells attached to rectal segments than to the other intestinal segments. In vitro, V. anguillarum cells did not adhere specifically to intestinal mucus, but rather accumulated close to intestinal mucus interfaces and subsequently penetrated them. It is proposed that the intestinal ttact of tutbot is a portal of entry for V. anguillarum and that the cells penetrate the intestinal mucus overlaying the epithelial cells.