罗非鱼无乳链球菌LrrG蛋白的原核表达及免疫原性分析

LrrG蛋白是无乳链球菌较保守的表面蛋白之一。为获得罗非鱼源无乳链球菌LrrG蛋白并探讨其在罗非鱼体内的免疫原性,本实验根据GenBank中已报道的人源无乳链球菌LrrG基因序列,设计特异性引物,扩增获得罗非鱼源无乳链球菌的LrrG基因。分析表明,其ORF为2 361 bp,编码786个氨基酸,与人源无乳链球菌LrrG基因核苷酸序列的相似性高达98.48%。LrrG蛋白含有3个保守的LRR结构域,并可形成多个抗原表位。将LrrG基因片段克隆转入原核表达载体pET-32a(+),构建重组质粒pET-32a(+)/LrrG,E.coli BL21(DE3)22℃诱导表达6 h。SDS-PAGE显示,诱导表达蛋白的分子量为108.9 ku,并且该重组蛋白以可溶和包涵体2种形式存在。经His Bind亲和柱纯化及超滤管浓缩后,LrrG可溶蛋白浓度达3.40 mg/mL。鱼体注射免疫实验表明,LrrG可溶蛋白对罗非鱼的相对免疫保护率达69.28%,且免疫后4周的血清抗体滴度为1∶800。该研究为深入探讨无乳链球菌LrrG蛋白作为罗非鱼基因工程疫苗的潜在应用价值奠定了基础。     

吉富罗非鱼sIgM基因多克隆抗体的制备及组织定位分析

为研究sIgM在吉富罗非鱼体内的组织分布及其在亚细胞水平上的定位,本实验利用纯化的SIGM融合蛋白,按照常规方法免疫新西兰大白兔,制备了兔抗吉富罗非鱼SIGM多克隆抗体;并对吉富罗非鱼肠、脾脏及鳃组织进行了免疫组织化学分析以及胶体金免疫电镜分析。结果显示,ELISA检测所获得的抗血清效价为1∶256 000,该血清能与SIGM融合蛋白发生特异性免疫反应。在肠和鳃组织中,阳性信号存在于富含黏液细胞的上皮细胞层表面,在杯状细胞和黏液细胞中则不存在;在脾脏组织中,阳性信号存在于特定的细胞中。免疫电镜结果显示SIGM主要存在于肠上皮细胞膜附近,并且在肠组织上皮细胞膜表面的微绒毛处含量较多;在鳃上皮组织中,SIGM主要存在于包围鳃丝和鳃小片的上皮细胞及部分红细胞内;而在脾脏组织淋巴细胞内部高尔基体的分泌小泡处存在大量的胶体金颗粒。研究表明,sIgM在鱼体黏膜免疫中具有重要作用,这为探讨鱼类sIgM的特性及功能奠定了基础,并丰富了鱼类黏膜免疫的研究内容。     

5种乳酸菌对奥尼罗非鱼免疫和抗病力的影响

选用体质量约3．49 g的奥尼罗非鱼,分别投喂基础饲料（空白组）和添加相同水平的嗜酸乳杆菌、植物乳杆菌、戊糖乳杆菌、鼠李糖乳杆菌、粪肠球菌的试验饲料。饲料中的乳酸菌数分别为1．30×103、5．10×108、2．97×108、3．03×108、2．30×108、3．47×108 cfu／g ,养殖期为8周,试验结束后采集血液并留部分鱼进行72 h的嗜水气单胞菌感染试验,计算72 h内的存活率。结果显示,植物乳杆菌组、戊糖乳杆菌组、鼠李糖乳杆菌和粪肠球菌组血清碱性磷酸酶活性均有不同程度升高,其中戊糖乳杆菌组最高,且显著高于嗜酸乳杆菌组;鼠李糖乳杆菌和粪肠球菌组血清抗超氧阴离子自由基能力显著高于其他各组;鼠李糖乳杆菌组血清溶菌酶活性显著高于空白组。注射嗜水气单胞菌12 h后,各添加组罗非鱼存活率均高于空白组,24～48 h ,鼠李糖乳杆菌组和粪肠球菌组存活率显著高于空白组,72 h时鼠李糖乳杆菌组存活率显著高于空白组。饲料中添加乳酸菌能提高奥尼罗非鱼免疫、抗氧化能力及抗嗜水气单胞菌感染能力,其中鼠李糖乳杆菌和粪肠球菌效果显著。     

Redox dysregulation and bioaccumulation of copper in tilapia Oreocromis niloticus

The objective of this study was to evaluate the effect of two concentrations of copper (0.13 and 0.5 mg L^?1) on juveniles of tilapia, Oreocromis niloticus (Linnaeus), using two toxicological assays (I‐ copper exposure and II‐ copper exposure followed by recovery). Doses were taken from the liver of the test organisms to determine the concentrations of copper, reduced glutathione, activities of glutathione S‐transferase and catalase, and lipid peroxidation. Exposure to copper in both assays resulted in significant accumulation of this metal in the analysed tissue and in decreased concentration of reduced glutathione. There was no alteration in the activities of catalase and glutathione S‐transferase and in lipid peroxidation.     

Genetic improvement of Israeli blue (Jordan) tilapia,Oreochromis aureus (Steindachner), through selective breeding for harvest weight

Genetic parameters and selection responses were obtained for harvest body weight of blue tilapia (Oreochromis aureus) from data collected over three generations in a selected population. A total of 18 194 records representing 186 sires and 201 dams were used in the analysis. Within generation heritability estimates for harvest body weight ranged from 0.18 to 0.58. When data from more than one generation were included in the analysis, heritability estimates became more stable (0.33–0.40) and it was 0.33 when all data were included in the analysis. The common full‐sib effect accounted for 10% of the phenotypic variance in the full data set. Heritability for survival from stocking to harvest was estimated at 0.01 and 0.09 in actual units (fitting an animal model) and in the logit (sire model) scale respectively. The genetic correlation between harvest body weight and survival was 0.22 and not significantly different from zero. The total selection response for harvest body weight over the three generations of selection measured as the difference between least‐squares means of selected and control lines was 17.7%. The corresponding figure when response was measured as the difference between mean breeding values of selected and control lines was 19.6%. The average inbreeding coefficient was 0.003 after three generations of selection. These results indicate that there are good prospects for the genetic improvement of harvest body weight in blue tilapia.     

饲料中脂肪水平对吉富罗非鱼幼鱼成活率、肌肉成分及消化酶活性的影响

研究了吉富罗非鱼幼鱼的成活率、肌肉成分及消化酶活性与饲料中不同脂肪水平的关系。设置饲料中鱼油添加量为0%、2%、4%、6%、8%和15%共6个梯度组,其脂肪水平分别为1.73%、3.71%、5.69%、7.67%、9.64%和16.55%共3个重复,饲养90d。吉富罗非鱼饲养中成活率的范围在84.76%~99.05%之间,但是投喂16.55%饲料的吉富罗非鱼成活率显著低于3.71%脂肪组(P<0.05)。吉富罗非鱼摄食量变化范围为(0.94±0.04)~(1.34±0.02) g/d,当饲料中脂肪水平大于7.67%时其摄食量显著下降(P<0.05)。7.67%组的试验末鱼平均体重和体长显著大于1.73%及16.55%组(P<0.05)。随着饲料脂肪水平提高,吉富罗非鱼的肌肉脂肪含量上升,变化范围为2.29%~4.27%,水分、粗蛋白、粗灰分及磷含量均呈下降趋势;吉富罗非鱼胃和肠道中蛋白酶活性没有显著变化(P>0.05),前肠和中肠的脂肪酶活性显著下降(P<0.05),前肠中的淀粉酶活性显著下降(P<0.05)。试验证明,高脂肪水平的饲料导致吉富罗非鱼成活率下降,摄食量减少;饲料脂肪水平提高,吉富罗非鱼肌肉脂肪含量随之增加,胃和肠道中的蛋白酶活性变化较小,但是肠道中脂肪酶的活性受到了抑制。     

Evaluation of visible implant elastomer tags for pathogenesis research in Nile tilapia (Oreochromis niloticus)

Two different colours (red and green) of visible implant elastomer (VIE) were used in Nile tilapia (Oreochromis niloticus). The visibility, location and retention of the VIE tags was investigated and any adverse effects on fish survival and growth determined. The use of VIE tags for monitoring individual fish during a bacterial challenge with either Streptococcus agalactiae or S. iniae was also studied. The results showed that VIE treated fish were lighter but not shorter than the non‐tagged control fish and that tagging caused no mortality. The retention of tags was better at the base of pectoral fin followed by the nasal area, lower abdomen, upper abdomen and branchiostegal rays inside the operculum. During the bacterial challenge experiment individual animals could be easily identified using the VIE tags. In this preliminary study, VIE tagging appears suitable for Nile tilapia research, as with other fish species, and could be a novel method to identify individual animals during microbial pathogenesis studies.     

不同杂交罗非鱼F_1代耐寒性能的研究

以3种品系尼罗罗非鱼的雌鱼作为母本,奥利亚罗非鱼的雄鱼作为父本进行杂交配对,研究杂交罗非鱼F_1代的耐寒能力。应用室内人工降温方法,测定了杂交子代死亡率、累积存活率和半致死低温(LT_(50))等耐寒指标。结果显示,3种杂交罗非鱼的耐寒能力各不相同,埃奥鱼、湘奥鱼和吉奥鱼半致死低温分别为:8.63℃、8.23℃、7.60℃。当水温降到9.6℃时,埃奥鱼首先开始死亡,其死亡低温范围为9.6~7.5℃;湘奥鱼的死亡低温范围为9.4~6.7℃;吉奥鱼的死亡低温范围为8.9~6.3℃。通过对3种杂交罗非鱼的LT_(50)进行单因素方差分析,结果表明3种杂交罗非鱼中,吉奥鱼的耐寒能力相对较强,其LT_(50)与埃奥鱼、湘奥鱼相比差异显著(P0.05),而其他2种杂交罗非鱼的LT_(50)相比差异不显著(P0.05)。     

尼罗罗非鱼cyp17a2生物信息学和雌雄性腺表达差异分析

[目的]进一步研究尼罗罗非鱼cyp17a2的编码蛋白的P450c17-Ⅱ的生物学特征以及在性腺中的表达特点。[方法]根据NCBI网站上登录的尼罗罗非鱼的cyp17a2基因全编码序列,利用相关的生物信息学分析软件分析cyp17a2基因编码的P450c17-Ⅱ的理化性质和结构特点,并分析该基因在不同性腺中的表达。[结果]罗非鱼cyp17a2基因编码的多肽含有521个氨基酸,p I为8.09,脂溶指数为98.33,是不稳定蛋白。罗非鱼cyp17a2基因的二级结构以不规则卷曲所占比例最大,为58.16%,α-螺旋占22.07%。P450c17-Ⅱ是二次跨膜蛋白,有众多蛋白质翻译后修饰位点。在60日龄性腺中,cyp17a2在精巢中的表达量显著高于卵巢。[结论]cyp17a2在尼罗罗非鱼不同性腺中的差异表达和编码P450c17-Ⅱ的生物信息学分析为研究cyp17a2在繁殖过程中的作用奠定了基础。