斑点叉尾鮰病毒“自杀性”ＤＮＡ疫苗的构建及其体外表达检测

根据CCVORF6基因序列,设计合适的引物,扩增ORF6基因,分别将其克隆到"自杀性"DNA疫苗载体pS-FV与常规DNA疫苗载体pcDNA3.1(+)中,转化感受态细胞DH5α后提取质粒,构建斑点叉尾鮰(Ictalurus punc-tatus)"自杀性"DNA疫苗ps-ORF6与常规DNA疫苗pcd-ORF6,利用转化后的大肠杆菌菌液为模板进行PCR扩增、提取质粒酶切鉴定以及序列测定等方法证实重组质粒构建正确。将重组质粒转染人胚肾细胞(293T),间接免疫荧光试验表明ORF6均获得表达,但"自杀性"DNA疫苗的表达效果不如常规DNA疫苗,该研究为这2种疫苗进一步的鱼体试验奠定了基础。     

斑点叉尾鮰出血病病毒疫苗的研究

斑点叉尾鮰病毒性出血病能使斑点叉尾鮰大规模的死亡,主要是由疱疹病毒病引起的。本实验通过对病鱼组织进行捣碎,离心,灭菌,灭活等几个实验步骤之后制备出疫苗。将制备的疫苗再用斑点叉尾鮰进行安全与毒理试验。结果表明给体长10 cm左右的小鱼注射0.1mL的疫苗比较适合,保护率为70%。该研究为进一步深入开展对此病的防治提供参考,以提高斑点叉尾鮰的养殖生产率。     

斑点叉尾鮰病毒性疾病综述

综述了斑点叉尾鮰病毒病的流行情况、临床症状,论述了影响疾病爆发的因子、诊断、治疗、控制和预防措施。     

斑点叉尾鮰病毒性疾病综述

本文综述了斑点叉尾鮰主要的病毒性疾病的流行情况、临床症状、诊断和治疗措施等,以期为斑点叉尾鮰的养殖生产和疾病防治提供参考依据.     

斑点叉尾鮰人工繁殖实验

我们于1989年开始进行斑点叉尾鮰的人工繁殖试验研究，现将试验情况报告如下。     

斑点叉尾鮰出血病病毒疫苗的研究

斑点叉尾鮰病毒性出血病是严重困扰着斑点叉尾鮰养殖业发展的一大疾病,主要是由疱疹病毒病引起的。通过对病鱼组织进行捣碎、离心、灭菌、灭活等步骤之后制备出疫苗,将制备的疫苗再用斑点叉尾鮰进行安全与毒理试验。结果表明,给体长10cm左右的小鱼注射0．1mL的疫苗比较适合,保护率为70％。     

红色和黑色斑点叉尾鮰人工繁殖试验

通过人工控制手段,促使红色斑点叉尾鮰与黑色斑点叉尾鮰自行配对、产卵、收集受精卵集中孵化,获得浅黑色的鱼苗;要获得稳定的红鮰鱼苗,必须采取红色斑点叉尾鮰自交的方法。     

广西地区斑点叉尾鮰人工繁殖试验

从池塘养殖的成鱼中选择亲鱼，培育至3龄以上，自然受精、人工孵化。4月上旬获第1批受精卵，经4d室内孵化。出苗36万尾，培育至7～8cm 32．4万尾，成活率达90％。比湖北地区产苗时间提前1个多月。     

斑点叉尾鮰亲鱼的运输

2000年3月30日,将美国密西西比品系原种斑点叉尾鮰(Ictalurus punctalus)亲鱼,自湖北荆州张家山渔场运往辽宁铁岭三家子渔场.起运亲鱼698尾共1 087kg,经20 h到达目的地,成活率97.9%.     

斑点叉尾鮰的人工繁殖技术

阐述了斑点叉尾的人工繁殖技术，其中包括亲鱼池的条件、亲鱼的选择与培育、亲鱼的产卵以及鱼卵的孵化。     

Efficacy of a Live‐attenuated Edwardsiella ictaluri Oral Vaccine in Channel and Hybrid Catfish

This study evaluated the efficacy of an oral live‐attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish (ESC) in channel and hybrid catfish. The vaccine was delivered one time orally by feeding fish a diet coated with an attenuated E. ictaluri isolate at four doses to deliver between 4 × 10⁶ to 3.2 × 10⁷ viable vaccine cells/g wet feed. Thirty‐five days postvaccination, control and vaccinated fish were challenged with virulent E. ictaluri and mortality was examined for 30 d postchallenge. Mortality of nonvaccinated hybrids (85%) and nonvaccinated channel catfish (73%) was similar but significantly greater than all groups of vaccinated fish. In channel catfish, a trend toward increasing mortality with decreasing dose was observed. Mortality of channel catfish vaccinated with the lowest dose (26.6%) was significantly higher than fish vaccinated with the highest dose (14.1%) but similar to fish vaccinated at the intermediate doses (17.5 and 19.4%). In contrast, mortality of four doses of vaccinated hybrid catfish was similar and ranged between 10.4 and 14.0%. The data demonstrate that the attenuated E. ictaluri vaccine at all four doses tested is effective at reducing ESC‐related mortalities in hybrid and channel catfish.     

佐剂对DNA疫苗的影响

DNA疫苗是近年来发展较为迅速的一类生物制剂,它能诱导动物机体产生持久的体液免疫和细胞免疫,其在抗病毒、细菌和寄生虫的感染方面起到了重要作用。但与传统的灭活疫苗相比,其免疫效果并不理想。为了攻克DNA疫苗免疫效力低下的难题,一些学者已经研制了多种佐剂制品,以提高DNA疫苗对动物疾病免疫保护的能力。该文针对使用佐剂提高DNA疫苗免疫效力的最新概况做一综述。     

Construction and expression of DNA vaccine against reddish body iridovirus and evaluation of immune efficacy in turbot (Scophthalmus maximus)

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome (VRBS) and high mortality caused by piscine turbot reddish body iridovirus (TRBIV). TRBIV virus is an icosahedron‐like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein (MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1‐TRBIV‐MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT‐PCR. After injection into turbot (Scophthalmus maximus), the expression of the antigen gene was analysed using RT‐PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post‐vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon‐induced antiviral molecules were observed in the vaccinated fish 60 h post‐vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot.     

DNA疫苗在动物疾病预防中的研究进展

DNA疫苗的出现是疫苗研究史上重大事件,被誉为疫苗研究的第三次革命。DNA疫苗既能激发机体的细胞免疫,也能诱导特异性的体液免疫,在预防和治疗细菌、病毒、寄生虫等感染性疾病等方面具有广阔的应用前景。利用DNA疫苗预防动物疾病是DNA疫苗发展的一个重要方面,并且在一些重大动物疾病预防研究上取得许多突破。可以肯定,随着人们对DNA疫苗免疫机制深入理解以及提高其诱导特异性免疫应答水平经验的积累,DNA疫苗定会在预防动物各种疾病方面发挥巨大作用。     

虹鳟传染性造血器官坏死病核酸疫苗工程菌发酵工艺研究

急性传染性造血器官坏死病(infectious hematopoietic necrosis,IHN)严重威胁虹鳟Oncorhynchus mykiss养殖业,造成严重的经济损失,核酸疫苗已成为虹鳟IHN疾病预防的研究热点。本研究首先检测虹鳟IHN核酸疫苗工程菌的遗传稳定性,结果显示工程菌在连续培养30代后,仍能保持稳定的质粒拷贝数。质粒酶切结果表明:连续培养30代后的质粒依然保持良好的完整性。在筛选高拷贝工程菌的基础上,又研究核酸疫苗的高密度发酵培养基、补料及发酵时间。结果表明:最优的发酵培养基配方为:1.2%胰蛋白胨(Tryptone)、2.4%酵母提取物(Yeast Extract)、0.5%甘油、0.017 mol·L~(-1)KH_2PO_4、0.072 mol·L~(-1)K_2HPO_4;补料配方为:3%胰蛋白胨、1.5%酵母提取物、50%甘油,最佳发酵时间为16h。本研究可为虹鳟IHN核酸疫苗的规模化生产提供参考。     

Development,distribution and expression of a DNA vaccine against nodavirus in Asian Seabass,Lates calcarifier (Bloch, 1790)

Fish nodavirus (betanodavirus), a viral pathogen responsible for viral nervous necrosis (VNN) was isolated from infected Asian sea bass (Lates calcarifer). The distribution, clearance and expression of nodavirus vaccine, on the basis of DNA vaccine (pFNCPE42 DNA‐pcDNA3.1) construction, were analysed in tissues of the Asian seabass by PCR, RT‐PCR, ELISA and Immunohistochemistry. Fish immunized with a single intramuscular injection of 20 μg of the pFNCPE42‐DNA vaccine showed a significant increase in the serum antibody level in the 3rd week after vaccination, compared to control eukaryotic expression vector pcDNA3.1 vaccinated fish. Results from PCR studies indicated that the vaccine‐containing plasmids were distributed in heart, intestine, gill, muscle and liver 10 days after vaccination. Clearance of pFNCPE42‐DNA vaccine was studied at 10, 25, 50, 75 and 100 days of post vaccination (d p.v). At 100 days p.v. pFNCPE42‐DNA was cleared from muscle of vaccinated sea bass. In vitro and in vivo expression of fish nodavirus capsid protein gene (FNCP) was determined by fluorescent microscopy. Asian seabass was immunized with pFNCPE42‐DNA vaccine at a dose of 20 μg per fish and were challenged with betanodavirus by intramuscular injection. The vaccinated seabass was protected from nodaviral infection and 77.33% of relative percent survival (RPS) was recorded.     

黄河鲶基因组DNA提取与纯化方法的研究

从鲶肌肉、肝、肾、血中提取基因组DNA。经过0.8%琼脂糖凝胶电泳检测,DNA带型整齐。紫外分光光度仪测定基因组DNA的D260 nm/D280 nm达1.77~1.82,证明所提取基因组DNA质量较好,可用作聚合酶链式反应(PCR)的模板所需。     

黄颡鱼Pelteobagrus fulvidraco AMH基因的克隆鉴定及表达

为研究抗缪勒氏管激素AMH对黄颡鱼Pelteobagrus fulvidraco性别分化及生长发育的调控作用,克隆扩增了黄颡鱼AMH基因的全长序列(GenBank检索号MK310106),研究该基因的组织表达模式。结果表明:AMH基因的全长为3588bp,其开放阅读序列为1653bp,编码550个氨基酸,位于第2号染色体上(19371164~19374761bp)。同源性分析表明,与已发布的黄颡鱼Tachysurus fulvidraco的相似性最高,可达99.6%;与低眼无齿[鱼芒]Pangasianodon hypophthalmus及斑点叉尾鮰Ictalurus punctatus的相似性也很高,分别达75.8%与74.8%,而与哺乳动物相似性较低。系统进化树分析显示:黄颡鱼AMH可与斑点叉尾鮰、低眼无齿[鱼芒]等鲶形目物种聚集成簇。Real-TimePCR检测结果表明:AMH的mRNA在各组织中的表达量差异性极大,在性腺组织中表达量最高,其次是肝脏和脑;1龄精巢的表达量最高,2龄次之,而1、2龄雌鱼的表达量基本相近。AMH对黄颡鱼性腺的分化及生长发育有重要调控作用。     

Construction of KHV‐CJ ORF25 DNA vaccine and immune challenge test

KHV ORF25 fragments were cloned from Koi Herpes Virus‐CJ (KHV‐CJ) strains isolated in our laboratory. The amplified products were inserted into the eukaryotic expression vector pIRES‐neo, forming recombinant plasmid pIRES‐ORF25. The recombinant plasmid pIRES‐ORF25 at 1 μg per koi, 10 μg per koi and 50 μg per koi was intramuscularly injected into healthy kois, respectively. The results showed that the recombinant pIRES‐ORF25 could induce the production of specific antibodies in koi determined by indirect ELISA. The differences of immune effect between three doses were not significant (P > 0.05), but all of them could induce the production of neutralizing antibodies. The immune challenge test showed that the mortality of koi injected with PBS, blank pIRES‐neo vector and nothing was 90%, 92.5% and 85% at 25 days. While the mortalities of koi injected with eukaryotic expression plasmid pIRES‐ORF25 were 20%, 17.5% and 12.5%. Differences in comparison with the control group were highly significant (P < 0.01). Histopathological staining revealed that the tissues of the immunized koi did not change apparently. In conclusion, the DNA vaccine pIRES‐ORF25 construct could well protect koi against KHV and had the potential to be applied in practice.