【作者】 邓成钢；

【导师】 秦爱建； 刘岳龙；

【作者基本信息】 扬州大学 ， 预防兽医学， 2008， 硕士

【摘要】 干扰素(IFN)作为细胞因子超家族中的一员,是一类由特定诱生剂作用于细胞分泌的具有一定抗病毒、抗肿瘤以及免疫调节功能等活性的糖蛋白,分为I和II两个型。I型IFN包括α、β、ω等亚型,而II型IFN则只有γ一个型。其中,IFN-α由于具有广谱的抗病毒活性以及一定的免疫调节功能而逐渐成为研究的热点. IFN-α主要由白细胞产生,与IFN-β,IFN-ω作用于同一受体,能抑制病毒DNA和蛋白质合成,促进MHC I类分子提呈抗原。目前,犬病毒性疾病在全球范围内呈现出越来越严重的趋势,在对这些病毒病的防治中,由于还没有犬干扰素的产品问世,都是使用人干扰素来代替犬干扰素。自Himmler等人于1987年首先开始了犬α-干扰素基因的研究以来,一些国外学者利于大肠杆菌或杆状病毒等表达系统表达出了犬α-干扰素基因,并对其生物学活性进行了初步探讨,而国内则只有王艳等在大肠杆菌与巴斯德毕赤酵母中对犬α-干扰素基因1亚型进行了表达,还未见犬α-干扰素抗犬病毒性疾病方面的研究报道。本实验通过RT-PCR方法从毕格犬外周血淋巴细胞中克隆出了犬的α-干扰素基因1亚型,将PCR产物连接至pGEM-T easy载体,挑取酶切鉴定阳性质粒进行测序,测序结果表明此次克隆的犬α-干扰素基因1亚型同与GenBank上所发表的登录号为m28624的序列同源性为100%。然后把该基因克隆进杆状病毒转移载体pFastBacI,在杆状病毒表达系统中对其进行了表达,将sf9细胞感染的P3代毒的培养上清通过VSV-CEF检测系统检测证实获得了有活性的犬α-干扰素。同时,还对α-干扰素基因1亚型的生物学特性进行了初步研究:实验表明犬α-干扰素在CEF上对CVI988与NDV(F48E8)均没有抑制活性,在MDBK上对VSV也没有活性,但在CEF上对VSV有抑制活性,说明犬α-干扰素有明显的组织特异性;将犬α-干扰素处理F81细胞后用CPV感染进行微量细胞病变抑制试验,即使1/2稀释度的犬α-干扰素仍没有抑制CPV的增值。更多还原

【Abstract】 Interferon (IFN) is a multigene family of inducible cytokines. IFNs which are commonly grouped into two types, generated by specific cells induced by inducer, are a kind of glycoprotein that has anti-viral activity, anti-tumor activity and immunoregulation function on the homogenecity animal cells. IFN type I is known as viral IFN and includes IFN-α(leukocyte), IFN-β(fibroblast) and IFN-ω. IFN Type II is known as immune IFN (such as IFN-γ). At present, IFN-αreseach is hot, which is synthesized mainly by Leukocytes and has a common receptor with beta, omega of IFNs. IFN–αcan inhibit virus replication, viral protein synthesis and promote MHC class I molecules presenting antigen.CaIFN-α1 gene was amplified by RT-PCR from the peripheral blood lymphocytes of beagle dogs. The PCR product was linked to pGEM-T vector and transformed into E. coli DH5α. The positive clones were screened by restriction enzyme digestion and sequenced. The results showed that CaIFN-α1 gene was 100% homology with that published in GenBank (No.M28624). Then CaIFN-α1 gene was cloned into the eukaryotic expression vector pFastBacI through a series of restriction enzyme digestion and ligation reactions. The positive recombinant plasmids were screened and identified by PCR. At the last, CaIFN-α1 was expressed in Sf9 insect cells. Supernatant of the cells infected with the recombinant virus could inhibit the cyto-pathic effect (CPE) in CEF infected with vesicular stoma-titis new jersey virus (VSV) . This result indicated that the CaIFN-α1 expressed in Sf9 cells has interfer activity to virus repilcation. However it could not inhibit CPE in CEF infected with CVI988 or NDV(F48E8). Also same result was found in MDBK cells infected with VSV or CPV- F81.更多还原