【作者】 王莉；

【导师】 夏春； 张鹤晓；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 随着斑马鱼全基因组测序工作的完成以及对EST研究的深入,斑马鱼干扰素基因于2003年2月被克隆报道,首次对鱼类干扰素基因进行了探索。为推进鱼类基因工程重组干扰素的研究,促进干扰素在水产业的应用,本文克隆并表达了我国重要经济鱼种—草鱼干扰素-α（GcIFN-α）基因,并进一步研究了重组GcIFN-α（rGcIFN-α）抗弹状病毒的效果。 首先构建了cDNA文库,根据斑马鱼、大西洋鲑和虹鳟干扰素序列设计一对兼并引物,再采用PCR技术克隆了一段草鱼干扰素基因。随后,设计了一对表达引物,克隆了草鱼干扰素成熟肽基因（mGIFN-α）。将mGIFN-α插入原核表达载体pQE30,经筛选、测序鉴定后,再将pQE30/mGIFN-α导入大肠杆菌JM109,进行了诱导表达。重组蛋白包涵体经离心收集、超声破碎、变性、洗涤、溶解、透析和复性后获得了纯化的GcIFN-α。最后,用细胞病变抑制法(CPE₅₀)分别测定了rGcIFN-α在草鱼卵巢细胞系（CO）、鲤鱼上皮瘤细胞系（EPC）、鸡胚成纤维细胞（CEF）上抗传染性造血器官坏死病毒（IHNV）、鲤春病毒（SVC）和滤泡性口炎病毒（VSV）的活性。 结果显示GcIFN-α成熟肽基因长474bp,编码158个氨基酸,在12～14位有一糖基化位点,在1位和97位存在两个半胱氨酸。GcIFN-α与斑马鱼、大西洋鲑、鲶鱼、金鱼干扰素比较,其氨基酸同源性分别为72.7%、46.8%、38.9%和99.3%。SDS-PAGE结果显示JM109（pQE30/GcIFN-α）工程菌株成功表达了分子量为20.2kDa、N端含有6个组氨酸的rGcIFN-α;表达的rGcIFN-α占菌体蛋白总量的42%;纯化后的rGcIFN-α电泳纯度大于90%。三次抗病毒实验结果表明rGcIFN-α在EPC/IHNV、CO/SVC系上具有明显的抗病毒活性,效价为1×10⁶～1×10⁷IU/mg,但在非鱼类细胞系CEF/VSV上无抗病毒活性。 本研究阐明了草鱼干扰素基因序列,并率先采用原核表达系获得了鱼类重组IFN-α,进行了rGcIFN-α抗IHNV、SVC效果测定,为rGcIFN-α在临床上的应用提供了可靠数据。更多还原

【Abstract】 After sequencing the zebrafish genome and along with the further researching on the expressed sequence tag (EST), the interferon of zebrfish was cloned in 2003 .To further evaluate the clinical impact of fish recombinant interferon, the grasscarp (C. idellus) IFN-α (GcIFN-α) gene was cloned from the cDNA library by PCR, and expressed in Escherichia coli expression system and assayed the anti-viral activity in the study.A pair of degenerate primers were designed based on conserved regions in fish interferon gene in DDBJ/GenBank. After amplification from cDNA library, the PCR product was inserted into pGEM T-easy vector and sequenced. Then, a pair of primers to express GcIFN-a was designed. The PCR product cloned by a standard PCR was subcloned into E.coli expression vector pQE30. The cells were harvested and total E.coli proteins were detected by SDS-PAGE to select expression strains. The rGcIFN-a was centrifuged, sonicated, then its renaturated and its effects on vesicular stomatitis virus, spring viremia of carp virus, and infectious hematopoietic necrosis virus were surveyed in homologous and heterologous cells by inhibition of the cytopathic effect.The cloned GcIFN-a gene encoded 158 amino acids, and had a potential N-glycosylation site and one pair of cysteines to form an intrachain disulfide bridge within site 1 and site 97. Comparison of GcIFN-a amino acids with other fish Type I IFN showed the identical ratio to be 30.6%-72.2%. The recombinant protein with 6 consecutive histidine residues was expressed with molecular weight of 20.2kDa. The proportion of rGcIFN-a in total E.coli proteins is approximately 42%. The anti-virus assay suggested that rGcIFN-a could inhibit these pathogenic rhabdoviruses on C. idellus ovaries and Epithelioma papulosum cyprini cell lines, but had no anti-VSV activity in chicken embryo fibroblast.更多还原