病毒铺膜印迹技术分离牙鲆鳃细胞上的淋巴囊肿病毒结合蛋白

利用非变性电泳与病毒铺膜印迹技术(VOPBA)分离了牙鲆(Paralichthys olivaceus)鳃细胞(FG)上淋巴囊肿病毒结合蛋白,结果显示在FG细胞膜上有分子量为135 kD的蛋白与淋巴囊肿病毒特异结合;对该蛋白切胶回收后进行SDS-PAGE与双向电泳,发现135 kD蛋白由3个蛋白组成,分子量分别为58.3 kD、44.6 kD及37.6 kD;135 kD蛋白SDS-PAGE的VOPBA显示,仅出现37.6 kD的蛋白带,而58.3 kD、44.6 kD蛋白皆不与淋巴囊肿病毒结合。结果表明牙鲆FG细胞上135 kD蛋白是淋巴囊肿病毒的结合蛋白,其37.6 kD蛋白具有病毒结合活性。     

淋巴囊肿病毒结构蛋白及其抗原性分析

病鱼为威海水产养殖场感染淋巴囊肿病的牙鲆（Paralichthys olivaceus）,收集病鱼的囊肿组织,匀浆破碎,采用差速离心和蔗糖密度梯度离心方法,分离纯化淋巴囊肿病毒粒子.负染后,电镜观察证实获得的病毒纯度高,杂质极少,病毒粒子呈近似于圆形的多角形,结构完整.纯化的淋巴囊肿病毒粒子经SDS-PAGE,硝酸银染色后,电泳图谱清晰显示病毒结构蛋白带共有22条,且分子量主要集中在123～26 kD.应用Western blotting法分析病毒结构蛋白的抗原性,结果显示,分子量分别为123.55 kD、65.292 kD和54.438 kD的3条蛋白带发生了免疫反应,其中分子量为65.292 kD的蛋白带反应强度明显高于其他2条蛋白带.本研究旨为确定淋巴囊肿病毒主要衣壳蛋白提供基础依据.[中国水产科学,2006,13（3）：415-420]     

牙鲆淋巴囊肿病毒纯化及不同来源病毒免疫特性比较

比较了3种不同的牙鲆淋巴囊肿病毒纯化方法,优化后的方法如下：剥离囊肿表面薄膜,收集内容物,匀浆后再用超声波细胞破碎仪破碎,反复冻融,650×g、1800×g差速离心,30%（W/W）蔗糖垫底超速离心（78500×g）浓缩病毒,最后蔗糖密度梯度超速离心（78500×g）纯化病毒。电镜观察发现,出现在47%～52%蔗糖密度区域的病毒带含有多量、纯净和结构一致的病毒粒子。此外,利用制备的兔抗血清对不同地区的病毒进行了免疫特性分析,Western blotting检测显示来自威海、青岛及秦皇岛3个地区的淋巴囊肿病毒反应结果是一致的,均有3条蛋白带发生反应,其分子量分别为125、66和55kDa。     

副溶血弧菌的外膜蛋白及其抗原性研究↑（*）

本文对１３株不同来源的副溶血弧菌的外膜蛋白及其抗原性进行了比较研究。１３株副溶血弧菌的外膜蛋白有相似的ＳＤＳ－ＰＡＧＥ图谱。大多数菌株有五条主要的蛋白质，其大致分子量分别为：ａ、６９ｋＤａ；ｂ、６３ｋＤａ；ｃ、４０ｋＤａ；ｄ、３０ｋＤａ；ｅ、２８ｋＤａ。其中蛋白质ｂ是所有菌株共有的。与吸附后的兔抗副溶血弧菌血清Ｗｅｓｔｅｒｎ印迹法结果显示抗血清与多数副溶血弧菌菌株的外膜蛋白有一条共同的免疫反应带，其大致分子量为４４ｋＤａ。     

南美白对虾人工感染细菌后肝胰脏中主要变化蛋白的研究

以采自汕头的南美白对虾为研究对象,运用病原菌人工感染、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、基质辅助激光解吸电离飞行时间质谱分析(MALDI-TOF/MS)、生物信息学和免疫印迹(Western-blot ting)等现代分子生物学方法,探索和鉴定对虾感染后肝胰脏中的主要变化蛋白。结果表明,南美白对虾人工感染溶藻酸弧菌6h之后,实验组和对照组肝胰脏的SDS-PAGE图谱出现12条明显差异条带,其中分子量分别为75kDa(命名为p75)和73kDa(命名为p73)的两种蛋白变化最为明显,进一步研究显示,p75和p73分别为血蓝蛋白的两个不同的亚基。提示,肝胰脏中的血蓝蛋白可能在对虾抗感染免疫中发挥重要的作用。     

淋巴囊肿病毒27.8 ku受体蛋白在牙鲆组织中的分布

应用抗牙鲆淋巴囊肿病毒(lymphocystis disease virus,LCDV)受体蛋白(27.8 ku)的单克隆抗体(2G11和3D9)定位LCDV受体蛋白在牙鲆组织中的分布。通过对牙鲆外周血、白细胞、鳃、胃、肠、表皮、肝脏、头肾、体肾、脾、性腺、脑、心脏等进行LCDV受体蛋白的间接免疫荧光与免疫组织化学定位观察,发现在牙鲆外周血白细胞的细胞膜、鳃上皮细胞、表皮、胃黏膜上皮细胞顶端、肠上皮细胞、肝细胞、脾表层结缔组织细胞及头肾后端的肾小管上皮细胞内均有较强的阳性信号,表明这些部位分布有LCDV的27.8 ku受体蛋白,但在体肾、性腺、脑、心脏及外周血红细胞中未观察到阳性信号。推测LCDV通过与鳃、表皮及消化道上皮的受体结合进入牙鲆体内,通过与外周血白细胞上的受体结合侵染白细胞而进入血液循环,进而感染肝脏、脾脏、头肾等器官。     

Isolation of lymphocystis disease virus from sole, Solea senegalensis Kaup, and blackspot sea bream, Pagellus bogaraveo (Brunnich)

Two viruses were isolated from cultured sole, Solea senegalensis, and wild blackspot sea bream, Pagellus bogaraveo, and preliminarily characterized as lymphocystis disease viruses (LCDVs). Viral isolates were characterized by morphological, biochemical and biophysical properties. In addition, the susceptibility of four fish cell lines was also tested. LCDV isolates developed cytopathic effects on the SAF-1 cell line at 5 and 6 days post-infection and reached titres of 10(6) TCID50 mL(-1). The antigenic and structural protein analysis of the two new LCDV isolates showed identical profiles to that obtained for LCDV strain Leetown NFH (ATCC VR-342), used as a reference viral strain, and for an LCDV isolate collected from gilt-head sea bream, Sparus aurata, cultured in southern Spain. Molecular confirmation was performed by polymerase chain reaction. Specific primers for LCDV produced a 270-bp DNA fragment, the expected size for LCDV.     

牙鲆淋巴囊肿病毒一抗原蛋白的确认及定位

以牙鲆淋巴囊肿病毒（LCDV）为抗原免疫Balb/c小鼠，而后将小鼠脾细胞与P3U1骨髓瘤细胞融合，以囊肿组织冰冻切片的免疫荧光染色筛选杂交瘤细胞，阳性结果显示特异性块状荧光信号集中在囊肿细胞的细胞质边缘部分，且多个荧光信号相连呈现链圈状，有限稀释 法克隆阳性杂交瘤细胞，三次克隆后获得4株稳定产生抗LCDV抗体的单克隆杂交瘤细胞株（1A8、1D7、2B6、2D11）。应用Western-blotting法分析单抗识别蛋白的分子量，结果显示，单抗1D7 和2B6均能特异性结合一条分子量116 kD病毒多肽；应用免疫电镜技术定位单抗识别的抗原决定簇，结果发现胶体金颗粒集中吸附在病毒粒子衣壳周围，且背景清洁，无散在的金颗粒或其他污染物。实验结果说明分子量约为116 kD的蛋白多肽为LCDV病毒衣壳蛋白，且具有线性抗原决定簇。     

Identification of novel immunogenic proteins of Vibrio alginolyticus by immunoproteomic methodologies

Vibrio alginolyticus is one of the most serious diseases in cultured marine and freshwater fish and shellfish. The absence of suitable vaccine or virulent marker can be the bottleneck to control V. alginolyticus infection. In this study, immunoproteomic approaches were undertaken to study the immunogenicity of the whole‐cell protein of V. alginolyticus HY9901. The whole‐cell proteins were analysed by two‐dimensional gel electrophoresis and subsequent immunoblotting using the rabbit anti‐V. alginolyticus HY9901 serum. A total of 55 immunogenic proteins were identified by immunoproteomic analysis. Of the 55 proteins, 51 are specific immunoreactive proteins and four are nonspecific immunoreactive proteins. Furthermore, outer membrane protein N (spot 2) was used as immunogens to immunize Epinephelus coioides for investigation of protective abilities and activities. The E. coioides immunized with OmpN has abilities to fight against infections caused by V. alginolyticus. The other novel immunogenic proteins may be developed as alternative antigens for further study of V. alginolyticus vaccine and diagnostics. These data show that immunoproteomics methods can be successfully applied in identifying immunogenic proteins of V. alginolyticus, which helps to search for the protective antigens in future.     

胶体金免疫层析法快速检测水产品中呋喃妥因代谢物残留

采用胶体金免疫层析技术(GICA)测定水产品中的呋喃妥因代谢物残留,并用液相色谱-串联质谱(LC-MS/MS)对实验结果进行确证。结果显示,胶体金免疫层析法的检测限为1.0μg/kg,假阳性率不大于5%,假阴性率为0;对实际样本的检测结果与LC-MS/MS法一致。研究表明,胶体金试纸条法操作简便、快速,成本低,为水产品中呋喃妥因代谢物的残留检测提供了快速筛选方法,而LC-MS/MS法灵敏度高,结果准确,适用于阳性样本的确证和精确定量。     

Protection against heterologous Streptococcus iniae isolates using a modified bacterin vaccine in Nile tilapia,Oreochromis niloticus (L.)

Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 μL of the vaccine or sham vaccinated with 100 μL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2–3 × 10⁷ CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS–PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22–35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.     

Phylogenetic analysis and molecular methods for the detection of lymphocystis disease virus from yellow perch, Perca flavescens (Mitchell)

Lymphocystis disease is a prevalent, non-fatal disease that affects many teleost fish and is caused by the DNA virus lymphocystis disease virus (LCDV). Lymphocystis-like lesions have been observed in yellow perch, Perca flavescens (Mitchell), in lakes in northern Alberta, Canada. In an effort to confirm the identity of the virus causing these lesions, DNA was extracted from these lesions and PCR with genotype generic LCDV primers specific to the major capsid protein (MCP) gene was performed. A 1357-base pair nucleotide sequence corresponding to a peptide length of 452 amino acids of the MCP gene was sequenced, confirming the lesions as being lymphocystis disease lesions. Phylogenetic analysis of the generated amino acid sequence revealed the perch LCDV isolate to be a distinct and novel genotype. From the obtained sequence, a real-time PCR identification method was developed using fluorgenic LUX primers. The identification method was used to detect the presence/absence of LCDV in yellow perch from two lakes, one where lymphocystis disease was observed to occur and the other where the disease had not been observed. All samples of fin, spleen and liver tested negative for LCDV in the lake where lymphocystis disease had not been observed. The second lake had a 2.6% incidence of LCD, and virus was detected in tissue samples from all individuals tested regardless of whether they were expressing the disease or not. However, estimated viral copy number in spleen and liver of symptomatic perch was four orders of magnitude higher than that in asymptomatic perch.     

Induction of apoptosis in a flounder gill cell line by lymphocystis disease virus infection

Lymphocystis disease virus (LCDV), a large icosahedral DNA virus classified to the iridovirus family, is the causative agent of lymphocystis, a disease which occurs in marine and freshwater fish species and is characterized by formation of papilloma-like lesions on the surface of the skin. In vitro, LCDV infection causes flounder gill cells, an adherent cell line, to exhibit an obvious cytopathic effect (CPE). In order to test whether apoptosis is responsible for the observed CPE, cells infected with LCDV at a multiplicity of infection (m.o.i.) of 5 PFU per cell were examined at various time intervals for the appearance of apoptotic signs. Nuclear fragmentation, DNA laddering and caspase activation were observed in the infected cells at the time (i.e. 10 days post-infection) when an intensive CPE was observed. These findings demonstrate that LCDV is capable of inducing apoptosis in vitro, which is different from the result of LCDV infection in vivo, and consequently suggest an intricate LCDV-host interaction.     

南极磷虾主要过敏原的分离纯化及性质研究

为了探究南极磷虾致敏问题,从南极磷虾中筛选、鉴定、分离纯化其主要过敏原,并对过敏原的性质进行研究,实验通过缓冲盐溶液提取南极磷虾蛋白;采用SDS-PAGE和Western blot(WB)筛选南极磷虾过敏原;使用高效液相色谱-串联质谱鉴定过敏蛋白;通过等电点沉淀、硫酸铵盐析、阴离子交换柱层析分离纯化过敏原;采用SDS-...     

鲤春病毒血症病毒糖蛋白的截短表达及其免疫原性分析

为了揭示鲤春病毒血症病毒(spring viremia of carp virus,SVCV)糖蛋白编码基因的主要免疫原性决定区域,本研究对SVCV的糖蛋白基因进行截短表达,并用纯化的重组蛋白作为免疫原制备了兔抗血清以分析其免疫原性。通过SOSUI以及DNAStar 6.0软件对SVCV糖蛋白基因的跨膜区、亲水性以及抗原表位进行分析后,采用RT-PCR对该基因主要抗原决定区域编码片段进行扩增,并构建重组表达质粒p GEX-Gtr,对其进行诱导表达后获得截短的SVCV糖蛋白的重组蛋白。将表达的重组蛋白进行纯化复性后,作为免疫原制备兔抗血清,采用ELISA法检测其效价,采用免疫印迹以及间接免疫荧光技术检测该重组蛋白的免疫原性。研究结果显示,截短表达的糖蛋白编码基因长1317 bp,编码439个氨基酸(29~467),推测分子量为49.6 k D。利用该重组蛋白制备的兔抗血清,其与重组蛋白的反应效价为1∶64000,。免疫印迹及间接免疫荧光结果显示,该兔抗血清与SVCV-HN株能发生特异性反应,表明该重组蛋白与天然的病毒表面糖蛋白的免疫原性无差异。上述研究结果表明,截短表达的重组蛋白具有很好的免疫原性,可应用于免疫诊断技术研发与基因工程疫苗的研制。     

Establishment of a novel fin cell line from Brown-marbled grouper, Epinephelus fuscoguttatus (Forsskål), and evaluation of its viral susceptibility

To lay a solid foundation of in vitro investigations of fish viral diseases, cytotechnology and cytotoxicology, a novel fin cell line from brown-marbled grouper, Epinephelus fuscoguttatus , was established and its viral susceptibility was evaluated. The fin tissues, digested with hyaluronidase and collagenase II, were used to initiate primary culture at 24 °C by using 20% foetal bovine serum-Dulbecco's modified Eagle medium/F12 medium, which was further supplemented with carboxymethyl–chitooligosaccharide, basic fibroblast growth factor and insulin-like growth factor-I. The fibroblastic fin cells grew at a steady rate during subsequent subculture and had a population doubling time of 50.6 h at passage 60. The modal diploid chromosome number was 48. A brown-marbled grouper fin cell line (bmGF-1) has been established and subcultured to passage 75 by now. Viral susceptibilities revealed that typical cytopathic effects of bmGF-1 cells emerged after being infected by turbot reddish-body iridovirus (TRBIV) or lymphocystis disease virus (LCDV). However, a large number of TRBIV and LCDV particles were also found in infected bmGF-1 cells. All these indicate that the bmGF-1 cell line has good susceptibility to TRBIV and LCDV, which may serve as a valuable tool for studies of cell–virus interactions and have potential applications in fish virus propagation and vaccine development.     

Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)‐GN438A expresses VP2 gene‐encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co‐infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross‐protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV‐N438A‐ΔNV‐EGFP and rIHNV‐N438A‐ΔNV‐VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild‐type (wt) IHNV HLJ‐09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ‐09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV‐N438A‐ΔNV‐VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.     

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 °C for 60 days, to compare LCD-incidence. In the fish reared at 20 °C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10⁶ PCR-U mg⁻¹ tissue, were detected in the fins and skin of LCD-affected fish at 20 °C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 °C and 30 °C; however, a low level of LCDV (10³ PCR-U mg⁻¹ tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 °C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 °C.     

固相萃取-液相色谱串联质谱法同时测定养殖海水中17种喹诺酮类药物

建立了固相萃取-液相色谱串联质谱法(SPE-LC-MS/MS)同时检测养殖海水中17种喹诺酮类药物残留的分析方法。海水经酸化处理后,采用HLB固相萃取柱富集、净化目标化合物,通过对比水样在不同上样pH、淋洗液与洗脱液等条件下的回收率,以此对前处理方法进行优化。收集到的洗脱液经氮气吹干后用流动相定容至1 ml,待测。色谱流动相A相为0.1%甲酸水溶液,B相为乙腈,所有药物经梯度洗脱进行分离,在LC-MS/MS正离子模式的多反应监测模式下进行定性定量分析。17种化合物可以在10 min中内得到较好的分离,线性范围为1–200 ng/ml,线性相关系数均大于0.999,检出限均在2–10 ng/L范围内,定量限均在5–20 ng/L范围内。以空白海水为基质,在20、100、200 ng/L三个不同添加水平下采用内标法定量的加标回收率均在71.3%–125.0%范围内,相对标准偏差(RSD)为2.44%–12.27% (n=5)。采用该方法对黄海灵山湾近岸4个养殖场进行海水采集并检测,共检测出4种喹诺酮类药物,分别是恩诺沙星、氧氟沙星、诺氟沙星和环丙沙星,其中,恩诺沙星浓度最高。研究表明,该方法快速、可靠,适用于养殖海水中喹诺酮类药物的检测。