【作者】 薛淑群；

【导师】 孙中武；

【作者基本信息】 东北林业大学 ， 微生物， 2012， 博士

【摘要】 锦鲤疱疹病毒(Koi Herpesvirus, KHV),即鲤科疱疹病毒3(Cyprinid herpesvirus3, CyHV-3),是一种双链、线性DNA病毒。锦鲤疱疹病毒是近年来在锦鲤和普通鲤鱼中发现的一种新病毒,它传染性强,死亡率高,呈世界性分布和流行,给鲤鱼和锦鲤养殖业造成了很大的经济损失,被列为我国二类动物疾病。目前的流行病学研究表明,鲤疱疹病毒传播迅速,水温22-27℃时爆发,死亡率高达80%-100%。由于该病毒造成病鱼的死亡率极高,疫情难以控制,已引起各国的高度关注。本研究建立了锦鲤尾鳍细胞系,通过染色体数目、核型以及DNA含量等实验,发现体细胞和培养细胞无显著性差异,染色体数目和DNA含量符合比例关系,建立的锦鲤尾鳍细胞系已形成了稳定的遗传性状,命名为KF-h。将感染病毒的鲤鱼肾细胞悬液接种于KF-h细胞单层,出现典型的KHV CPE现象,通过电镜观察、PCR扩增和序列分析以及动物回归试验,表明已成功分离到KHV病毒,命名为KHV-hlj。锦鲤疱疹病毒KHV-hlj株经过灭活制成细胞灭活疫苗,鲤鱼经注射和浸泡免疫细胞灭活疫苗后,通过对其白细胞吞噬能力、淋巴细胞转化能力的检测以及攻毒试验证明,非特异性免疫、细胞免疫和体液免疫均显著提高；此外,这种制备方法能有效保留病毒致病性抗原的完整性,完整的病毒抗原有效地刺激鲤鱼产生较强的免疫反应,最终导致了受疫鲤产生较强的免疫保护力。锦鲤疱疹病毒KHV-hlj株,菌株自细胞系提取,KHV-hlj株细胞灭活疫苗,配方合理,操作简便,价格适中,适于批量生产,使疫苗的推广应用成为可能。疫苗的免疫结果表明,通过注射途径接种疫苗,比浸泡获得更高的凝聚抗体效价和免疫保护率。实验扩增了锦鲤疱疹病毒包膜糖蛋白gD基因,序列测定表明了包膜糖蛋白gD基因具有高度的遗传稳定性,核酸序列同源性达99.64%,主要功能区的序列未发生变化,对gD蛋白的功能和结构没有影响。锦鲤疱疹病毒KHV包膜糖蛋白gD基因的全长1179bp,编码393个氨基酸,25个疏水氨基酸的氨基末端信号肽,成熟肽只有368个氨基酸。KHV-gD蛋白是跨膜糖蛋白,跨膜螺旋区位于N端4-25位和C端341-363位之间,其大部分肽链位于膜外区,只有小部分在胞内。克隆的KHV-gD在去除了N端的前25个氨基酸(信号肽)后,为该蛋白的成熟肽,序列分析表明其有34个碱性氨基酸,35个酸性氨基酸,130个疏水氨基酸,92个极性氨基酸,分子量为40.63kD,等电点pI为7.15; KHV-gD成熟肽与KHV-gD相比,C端跨膜螺旋的结合分值有所升高(从2217升高至2777)。本研究构建了含完整KHV-g基因的巴斯德毕赤酵母表达载体pPIC9K-KHV-gD,在酵母菌中表达KHV-gD蛋白时,可保证KHV-gD免疫原性的完整,为研制重组蛋白诊断试剂盒及开发基因工程疫苗奠定了基础。此外,也为研究KHV-gD蛋白的结构和功能提供了有利条件。更多还原

【Abstract】 A new continuous cell line (KF-h) from caudal fin of koi, Cyprinus carpio koi, was developed and sub-cultured more than100passages since the present study was initiated.Cell line KF-h predominantly consisted of short fibroblast-like cells and grew well in Dulbecco’s modified Eagle medium (M199) supplemented with10%fetal bovine serum (FBS). Chromosome analysis revealed that56%of Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. Koi herpesvirus [KHV or cyprinid herpesvirus3(CyHV-3)] infections in carp are a serious problem worldwide. Since1988, KHV outbroke in various countries and caused serious economic loss therefore KHV became one of quarantine important projects. KHV was first isolated and confirmed in1998from diseased common carps and koi in Israel and the USA. Acomparison of virus characteristics reveals that KHV is a distinct virus species from two well-known piscine herpesviruses:cyprinid herpesvirus (CyHV-1) and channel catfish herpesvirus (IcHV-1). At the molecular level, multiple sequence alignment analysis indicates that CyHV-3belongs to a new species of cyprinid herpesvirus (the third cyprinid herpesvirus, CyHV-3) of the family Herpesvirus,。KHV has become a globally distributed pathogen, and has been considered one of the most important causative agents to koi and common carps worldwide.Through the experiments of the chromosome number, karyotype and DNA content, the cell line of the koi’s tail fin established in this study demonstrates that there is no obvious difference between the somatic cells and culture cells; the chromosome number and the DNA content are in accordance with the proportionate relationship; and the established cell line of the koi’s tail fin, named as KF-h, has formed the stable heredity.When the nephrocyte suspension of the carp with the typical symptoms is inoculated to the KF-h cell monolayer through the observation of electron microscope, PCR amplification, sequence analysis and the animal regression experiment, the appearance of the typical KHV CPE phenomenon will indicate that the KHV virus has been separated successfully and named as KHV-hlj.The KHV-hlj strain is made into the cell inactivated vaccine through inactivation, and after the carp is injected and immersed with the immunocyte inactivated vaccine, the detection and virus attacking test towards the leukocyte phagocytic activity and the lymphocyte transformation ability of the immunized carp have initially proven that the carp has the impeccable immune competence, and the non-specific immunity, cellular immunity and humoral immunity can be improved remarkably with vaccination; this test also proves that this preparation method can effectively keep the pathogenic antigen of the virus; the antigen of the virus can effectively stimulate the carp to produce the strong immune reaction, which finally leads to the strong immune protection created by the immunized carp. As for the cell inactivated vaccine of the KHV-hlj strain, the bacterial strain is collected from the cell line with the reasonable formula, easy operation and moderate cost, so it is suitable for the volume production, which enables the application and dissemination of the vaccine to become possible. The immune result of the vaccine demonstrates that if the carp accepts the vaccination by means of injection instead of immersion, the higher agglomerate antibody titer and the immune protection rate will be achieved.The experiment has augmented the mature peptide of the KHV-hlj envelope glycoprotein D gene, and the sequence determination has indicated that gD gene has the high genetic stability, with the nucleic acid xequence homology reaching99.64%; besides, the sequence of the main functional zone doesn’t change and make no difference to the function and structure of gD protein. The overall length of the envelope glycoprotein coding gene of KHV is1179bp, and it is coded with US6gene; the amino-terminal signal peptides of393amino acids and25hydrophobic amino acids have been coded, and the mature peptide only has368amino acids, with the transmembrane region located in the place of No.341-363amino acids. The KHV-gD protein belongs to the transmembrane glycoprotein, with only a little part inside the cell, and the transmembrane spiral area of this protein is located at No.4-25of N end and No.341-363of C end, the part between which belongs to the envelope region. The cloned KHV-gD polypeptide has removed the transmembrane region of the first25amino acids of N end (signal peptide) and become the mature peptide of this protein, and its combined scores of the transmembrane helix at C end has increased from2217to2777. When analyzing the sequence of the cloned KHV-gD mature peptide, we can find that there are368amino acids in total with the molecular weight as40.63kD and the isoelecric point p1as7.15, including34basic amino acids,35acidic amino acids,130hydrophobic amino acids and92polar amino acids.This study has constructed the Pichia pastoris expression vector pPIC9K-KHV-gD which contains the complete mature peptide gene of KHV-gD. When further expressing the KHV-gD protein in the saccharomycetes, we can ensure the integrity of the KHV-gD immunogenicity, so as to lay the foundation for studying the diagnostic kit of the recombinant protein and develop the vaccine of the genetic engineering. Besides, the expression of KHV-gD protein in the Pichia pastoris also provides the advantage for researching the structure and function of KHV-gD protein.Because KHV causes severe financial losses in the common carp and koi culture industries worldwide, it is a useful subject for applied science. Safe and efficacious vaccines adapted to mass vaccination of carp and efficient diagnostic methods need to be developed. Several aspects of KHV make it also useful for fundamental science. These aspects are its large genome, the relationship between KHV infectivity and temperature, and the low similarity between KHV genes and the genes of other members of the order Herpesvirales that have been studied. Further studies are needed to identify the roles of KHV genes in viral entry, egress, and disease pathogenesis.更多还原