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。     

传染性造血器官坏死病毒抗原表位富集区的原核表达及应用

以传染性造血器官坏死病毒Sn1203株(IHNV-Sn1203)基因组RNA提取物为模板,利用生物信息学软件分析,通过RT-PCR一步法扩增截短的G蛋白基因序列(约375 bp),将其克隆到表达载体p ET-27b中,构建重组表达质粒p ET-27b-IHNV-short G,通过大肠杆菌Rosetta表达菌株获得高效表达。在IPTG浓度为0.25 mmol/L时,37℃诱导表达,经SDS-PAGE电泳分析显示目的蛋白相对分子质量约为14 000,符合预期大小,并以包涵体的形式表达,4 h时目的蛋白表达量最大。蛋白经变性、复性处理后获得不带任何标签的纯化蛋白,并利用该蛋白制备兔抗血清。ELISA结果显示,兔抗血清的效价为1∶80 000,说明制备的兔抗血清能够识别表达的重组蛋白;间接免疫荧光结果表明兔抗G蛋白血清具有良好的特异性,并且与VHSV参考毒株没有任何交叉反应。     

传染性造血器官坏死病毒糖蛋白抗血清的制备及应用

应用逆转录聚合酶链式反应(RT-PCR)从传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)感染的细胞悬液克隆病毒的糖蛋白基因,将其亚克隆至原核表达载体pCWori,转化到大肠杆菌DH 5α,通过发酵大肠杆菌制备病毒糖蛋白.经SDS-PAGE分析,诱导表达的重组蛋白主要以包涵体的形式存在,使用Ni-NTA亲和层析柱在变性条件下进行纯化并透析复性,最终得到了较高纯度的可溶性糖蛋白,分子量约为57 kDa.Western-blot分析结果显示,所表达的蛋白能够被IHNV病毒制备的兔抗IHNV血清识别.用复性后的蛋白免疫小鼠制备抗血清,ELISA显示抗体效价可达1∶64 000.经制备的抗血清可以作为一抗建立ELISA检测方法,用于检测细胞悬液的病毒粒子,将抗血清稀释到1∶16 000仍能与IHNV全病毒发生反应.本研究利用重组的IHNV糖蛋白成功制备了高效价的抗血清,并能够与IHNV全病毒发生特异性结合,为IHNV免疫学检测方法建立奠定了基础.     

传染性胰腺坏死病病毒分离株VP2基因抗原表位区融合表达及免疫特性的分析

利用RT-PCR方法扩增出IPNV-ZYX分离株主要结构蛋白VP2的抗原表位区基因(616 bp), 命名为IPNV VP2 COE, 将其克隆到pCold TF表达载体中构建重组质粒pCold TF-VP2 COE, 在大肠杆菌BL21(DH5α)感受态表达, 经SDS-PAGE电泳分析, 表达蛋白约78 ku, 用镍离子亲和层析柱纯化该蛋白, 制备抗血清, 间接ELISA结果显示, IPNV (ATCC VR-1318)细胞培养物与鼠抗VP2 COE蛋白血清发生特异性反应, 效价为1∶12 800; 间接免疫荧光结果显示, 鼠抗VP2 COE血清可与黑龙江某渔场已知感染IPNV虹鳟肝组织产生特异性的荧光, 以上两项结果表明, 表达IPNV VP2 COE蛋白具有良好的免疫原性和免疫反应性, 为IPNV检测方法的建立及疫苗的制备提供理论依据     

传染性胰腺坏死病毒VP3蛋白的原核表达及抗原性分析

应用RT-PCR方法扩增了IPNV编码内衣壳VP3蛋白的基因615bp,将VP3基因克隆至原核表达载体pET30b,并在大肠杆菌BL21中得到了表达。通过SDS-PAGE分析表明,重组菌诱导后得到了预期大小约30ku的VP3蛋白,与理论值相符,经薄层扫描分析表明目的蛋白表达量可占菌体总蛋白的30%。用镍离子亲和层析柱纯化可溶性的VP3蛋白,并制备抗血清。Western-blotting结果显示,VP3蛋白可被兔抗IPNV阳性血清识别;间接ELISA结果显示,IPNV细胞培养物作为抗原,兔抗VP3蛋白高免血清稀释度为1∶25600时,P/N>2,抗血清可与IPNV全病毒发生反应,以上两项结果说明,表达的VP3蛋白与天然的IPNVVP3蛋白一样具有相同的抗原性。试验利用原核表达系统成功地高效表达了IPNVVP3蛋白,融合蛋白以可溶性形式存在,并制备了高效价的抗血清。     

5株鲆鲽鱼类病原菌多克隆抗体的制备与应用

本研究制备了鳗弧菌Vibrio anguillarum、杀鲑气单胞菌Aeromonas salmonicida、副溶血弧菌V. parahaemolyticus、哈维氏弧菌V. harveyi和腐败希瓦氏菌Shewanella putrefaciens 5株鲆鲽鱼类病原菌的兔抗血清,建立了5种菌的间接ELISA检测方法,并将该检测方法用于鱼类细菌分离物病原检测。人工感染结果显示,5株菌对大菱鲆的半数致死量(LD50)在102~107CFU/fish;制备的兔抗血清效价分别为1∶2 048 000、1∶16 000、1∶16 000、1∶1 024 000、1∶128 000;交叉反应结果显示,鳗弧菌、副溶血弧菌、哈维氏弧菌3株弧菌与抗血清相互之间存在交叉反应;抗血清特异检测灵敏度分别为104、108、107、105、106cell/ml;对13株海水鱼类细菌分离物进行检测,有1株腐败希瓦氏菌阳性,两株哈维氏弧菌阳性;1株副溶血弧菌和哈维氏弧菌均为阳性,该结果与16S rDNA序列的分子分析方法一致。     

虹鳟IL-17多克隆抗体的制备及初步鉴定

本研究根据NCBI已发表序列设计引物,提取经植物血凝素刺激后的虹鳟Oncorhynchus mykiss头肾细胞总RNA,采用RT-PCR方法扩增IL-17成熟肽基因,测序结果表明所获得的序列与发表序列相一致。将该基因重组至原核表达载体pET32a中,并转化大肠杆菌Escherichia coli Rosetta,进行诱导表达。SDS-PAGE电泳结果表明:目的蛋白以包涵体形式表达,大小约为32k Da,重组蛋白经Ni-NTA系统纯化、复性后纯度达90%以上,以其免疫小鼠制备虹鳟IL-17多克隆抗体。ELISA结果显示其效价为1∶25 600,而间接免疫荧光结果显示所制备的多克隆抗体能够特异性地识别真核细胞中瞬时表达的虹鳟IL-17。本研究成功制备虹鳟IL-17多克隆抗体,为下一步IL-17在虹鳟黏膜免疫中的作用及其佐剂效应研究奠定基础。     

黄鳝血清转铁蛋白多克隆抗体的制备及检测

为实现黄鳝血清转铁蛋白基因的原核表达并制备其多克隆抗体,利用基因特异性引物从黄鳝肝脏cDNA中扩增黄鳝转铁蛋白的C端序列,亚克隆至原核表达载体pET-28a(+)中,构建pET/Tf-C重组表达载体;转化大肠杆菌BL21(DE3)后进行IPTG诱导。利用Ni离子亲和层析技术纯化Tf-C蛋白,并免疫新西兰兔制备多克隆抗体;通过间接ELISA技术和组织蛋白印迹对制备的多克隆抗体进行检测。试验结果表明,成功构建pET/Tf-C原核表达载体,并实现了蛋白的表达和纯化;制备的多克隆抗体效价大于1∶25 600,并能特异性地识别来源于黄鳝不同组织的血清转铁蛋白。研究结果对黄鳝血清转铁蛋白功能的研究奠定了基础。     

黄鳍棘鲷血清IgM的纯化及兔抗血清的制备

分别采用Sepharose-6B凝胶过滤层析、Phenyl Sepharose FF疏水作用层析和rProtein A Sepharose亲和层析技术纯化黄鳍棘鲷（Acanthopagrus latus）血清IgM,并将这3种层析纯化方法进行了比较。纯化产物进行SDS—PAGE,电泳结果扫描后经Band Scan5．0软件分析显示,单独使用Sepharose-6B凝胶过滤层析或Phenyl Sepharose FF疏水作用层析得到的IgM纯度只有56％左右;二者联合使用纯度可以达到69％,而rProtein A Sepharose亲和层析一步就可以达到89％的纯度;纯化得到的黄鳍棘鲷血清IgM的重链和轻链分子量分别为73．6kD和26．5kD。本研究还以rProtein A Sepharose亲和层析纯化的黄鳍棘鲷血清IgM为抗原,制备其兔抗血清,采用Western Blot和双向琼脂扩散检测抗血清免疫学活性,并用间接ELISA检测其效价达到1：25600,为进一步开展黄鳍棘鲷免疫学方面的研究奠定了基础。     

赤点石斑鱼神经坏死病毒主衣壳重组蛋白多克隆抗体的制备

把赤点石斑鱼（Epinephelus akaara）神经坏死病毒（RGNNV）主衣壳蛋白（MCP）基因的重组表达质粒载体pRSETA-MCP转化至大肠杆菌（Estherichia coli）BL21（DE3）,经IPTG诱导表达,SDS-PAGE显示表达的重组蛋白主要以不可溶的包涵体形式存在,分子量约44.5kD。通过Ni-NTA-Agarose亲和层析柱纯化,经分析纯度达90%,之后免疫新西兰兔制备抗血清,ELISA效价达1：12800以上。Western-blot分析结果显示,该血清与表达的重组蛋白有较强反应,说明通过原核表达的重组蛋白具有良好的免疫原性。     

Passive immunisation of fish against vibriosis, comparison of intraperitoneal, oral and immersion routes

Passive immunisation of fish was conducted to determine whether anti-Vibrio anguillarum whole sera (AVA) and affinity-purified AVA raised in sheep, rabbits and rainbow trout (Oncorhynchus mykiss) were persistent when injected and orally administered into rainbow trout. These responses were compared with active immunisation by immersion in, and intraperitoneal (i.p.) injection with, formalin-killed V. anguillarum cells. Sheep and rabbit AVA were detected in rainbow trout sera for up to 70 days (half-life 21 days) after i.p. injection as determined by an enzyme-linked immunosorbent assay (ELISA). The relative percentage survival (RPS) of passively immunised rainbow trout challenged with virulent V. anguillarum after an injection was comparable to that of active immunisation by immersion after 1 month post-immunisation (p.i). Affinity-purified sheep and rabbit AVA exhibited the same protective potential as whole serum in rainbow trout. Rabbit and sheep immune sera diluted 1:8 and 1:50, respectively, provided equivalent protection as undiluted fish immune serum. An active immune response against passively acquired heterologous immunoglobulins was demonstrated by ELISA, with responses against sheep AVA being less than those against rabbit AVA. Rainbow trout given purified sheep AVA conjugated to LTB (the GM-1-binding subunit of Escherichia coli heat-labile toxin) and administered orally had an RPS of 37.5% at 15 days and 27% at 1 month p.i. In contrast, fish given sheep AVA conjugated to TraT (an internal membrane of E. coli) or in micellar form with Quil-A had RPSs of only 18.7 and 6.2%, respectively, after 15 days, and 13.3 and 0% after 1 month, respectively. The protection conferred by immune sera was shown to be due to the immunoglobulin component alone. Heat inactivation of the complement in sera had no effect on the potency of immune sera.     

Production of biologically-active recombinant goldfish gonadotropins in transgenic rainbow trout

The feasibility of using rainbow trout Oncorhynchus mykiss embryos as an expression system for proteins was investigated. For model proteins, we selected two goldfish gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). To produce single-chain goldfish FSH (scgfFSH) and LH (scgfLH), cDNAs encoding glycoprotein hormone (GP) α and FSHβ were fused in tandem, and cDNAs encoding GPα and LHβ were fused in tandem. The fused cDNAs were ligated with β-actin promoter, and microinjected into fertilized rainbow trout eggs. After 4-days incubation, the embryos were subjected to western blotting and in vitro bioassays. The recombinant proteins produced by the embryos were immunoreactive to antisera against goldfish GPα, N-glycosylated, and biologically active. We conclude that scgfFSH and scgfLH were successfully produced in transgenic rainbow trout.     

Experimental evidence for direct in situ binding of IgM and IgT to early trophonts of Ichthyophthirius multifiliis (Fouquet) in the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Freshwater fish are able to mount a protective immune response against the parasite Ichthyophthirius multifiliis (Ich) following a non‐lethal exposure. Factors involved in immunity comprise cellular and humoral factors, but antibodies have been suggested to play a prominent role in protection. However, host antibodies have not yet been demonstrated to bind to the parasite in situ. By the use of immunohistochemical techniques, this study demonstrated that IgT and IgM bind to surface structures, including cilia, on the early feeding stage of the parasite in the gills of immune rainbow trout, Oncorhynchus mykiss, shortly (2 h) after invasion. No binding of IgT and no or only a weak binding of IgM was observed on the parasites in the gills of similarly exposed but naïve rainbow trout. This study indicates that antibodies play an important part in the protection of immune fish against Ich although additional humoral and cellular factors may contribute to this reaction.     

The Effect of Kefir as a Dietary Supplement on Nonspecific Immune Response and Disease Resistance in Juvenile Rainbow Trout,Oncorhynchus mykiss (Walbaum 1792)

In this study, juvenile rainbow trout fed with commercial pellets containing kefir provided increased nonspecific immune response and improved disease resistance against lactococcosis and yersiniosis. Kefir was used as a feed supplement at 2, 5, and 10% inclusion rates and several nonspecific immune parameters were observed at day(s) 1, 7, 14, 21, 28, and 35 following the treatment. A total of four experimental groups, including control, was established. The various parameters including hematocrits, nitroblue tetrazolium positive neutrophils, total leukocytes, serum lysozyme activity, total serum protein, and immunoglobulin M (IgM) levels were examined. As a result of this study, kefir‐fed fish had an increase in measured nonspecific immune parameters, especially in the group received the 10% kefir treatment. The challenged fish fed with kefir‐supplemented diet showed a better survival rate against Lactococcus garvieae than Yersinia ruckeri. Kefir supplementation reduced fish mortality significantly against L. garvieae.     

The non-specific activation of rainbow trout, Salmo gairdneri Richardson, complement by Aeromonas salmonicida extracellular products and the correlation of complement activity with the inactivation of lethal toxicity products

Abstract The possible mechanism of inactivation of the toxicity of Aeromonas salmonicida extracellular products (ECP) by normal rainbow trout serum was investigated using juvenile rainbow trout. ECP was prepared from culture supernatant by an acetone precipitation method. The ECP was incubated with normal rainbow trout serum at 20°C for 2 h, and the interrelationship between ECP proteolytic activity and immune complex-initiating, haemolytic complement activity (CH₅₀) of normal serum against antibody-sensitized goldfish red blood cells was evaluated. When normal serum was incubated with increasing concentrations of ECP, the CH₅₀ activity of serum decreased. The CH₅₀ activity was completely abolished in serum treated with undiluted ECP. ECP treated with serum was administered to trout intraperitoneally to determine mortality. All the fish receiving untreated ECP (0.05 ml = 0.5 mg protein) alone died within 24 h. When ECP was treated with serum at 1:1 to 4:1 (serum: ECP) in volume a similar high mortality was produced. These inocula possessed high protease activity and no or low CH₅₀ activity. However, mortality decreased and finally no mortality was recorded as ECP was treated with large volumes of serum (9:1 to 19:1). These inocula had lower protease activity and considerably higher CH₅₀ activity. Fish receiving ECP treated with heat-inactivated serum at 19:1 showed 100% mortality. A serum: ECP inoculum derived from fish which had been administered lipopolysaccharide from Salmonella enteritidis and which possessed a low CH₅₀ activity also gave a high mortality when used at 19:1. These results suggest that rainbow trout complement is implicated in the inactivation of toxicity of A. salmonicida ECP.     

Effects of Oral Administration of Specific Antibodies to Rainbow Trout Oncorhynchus mykiss

A new product called oralized fish serum concentrate (OFSC) was evaluated for a possible effect against various bacterial pathogens in rainbow trout. The OFSC produced from immune trout sera was found to contain fully functional antibodies and complement component C3. The antibodies detected in the serum concentrate were specific to Vibrio anguillarum (O1 and O2) and Aeromonas salmonicida , which had been used for vaccination of the fish prior to serum collection. The functionality of the specific antibodies in OFSC was not reduced after 6 wk storage at -20 C, 5 C, and 20 C. The serum was mixed with commercial trout feed and used for feeding rainbow trout fry (first feed period). After oral delivery of OFSC to rainbow trout for 1 mo, samples of gut content and gut tissue contained functional antibodies. In gutted fish no functional antibodies were found. This suggests that antibodies from OFSC are unable to be transferred across the gut wall in a functional state. Oral administration of OFSC did not increase survival of rainbow trout in an immersion challenge with Vibrio anguillarum .     

Humoral factors important in resistance of salmonid fish to bacterial disease. I. Serum antibody protection of rainbow trout (Salmo gairdneri) against vibriosis

Serum antibody is shown by passive immunization and absorption experiments to be an important component of defense mechanisms against experimentally induced vibriosis in rainbow trout (Salmo gairdneri). Antigen from heat-killed Vibrio anguillarum injected into trout with Freund's complete adjuvant induced much higher agglutinin titers than formalin-killed bacteria with or without adjuvant, or heat-killed bacteria without adjuvant. Passively transferred trout anti-Vibrio anguillarum serum provided protection against experimental vibriosis in trout for more than 2 months. Passively transferred anti-Vibrio anguillarum rabbit serum also protected trout.     

Gastric uptake of recombinant growth hormone in rainbow trout

The aim of the present study was to investigate gastrointestinal uptake of recombinant growth hormone in rainbow trout. Recombinant human GH was used in this study to avoid cross reactivity with the endogenous trout GH molecule. Using both in vitro and in vivo approach, the results demonstrate that stomach is the main site of uptake for orally administered intact recombinant human growth hormone (hGH) in rainbow trout. The presence of hGH was verified by western blot analysis in the stomach tissue as well as in the serum after 30 minutes of intubation. No traces of the hGH could be detected after 90 minutes of administration, indicating rapid clearance of the hormone in rainbow trout. Furthermore, no immunoreactive hGH was detected in other tissues including mid-gut, muscle and liver at the time points tested. The findings provide novel information on the gastric uptake of hGH in rainbow trout, and would be of relevance to the application of GH in aquaculture.