摘要
亚克隆犬α1干扰素(CaIFN-α1)成熟蛋白编码基因并克隆到表达载体pPICZα-A,构建转移重组载体pPICZα-A-CaIFN-α。pPICZα-A-CaIFN-α经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。转化子经PCR分析鉴定后利用甘油增菌和甲醇诱导,实现了CaIFN-α1在毕赤酵母系统中的分泌表达。SDS-PAGE检测结果表明表达产物相对分子质量约为2.7×104,比其推导结果(约1.9×104)大,推测可能是发生了糖基化。酵母分泌表达的CaIFN-α1具有较高的抗病毒活性,约为1.45×106U·mL-1,蛋白含量约为96mg·L-1,比活性为1.49×107U·mg-1。重组CaIFN-α1的生物学活性具有较强的种属特异性,在犬肾细胞(MDCK)上具有很高的抗病毒活性,在鸡胚成纤维细胞上活性较低,而在猫肾细胞(F81)和牛肾细胞(MDBK)上几乎没有抗病毒活性。进一步研究发现,重组犬α1干扰素对犬瘟热病毒和伪狂犬病毒在犬肾细胞中的增殖具有显著抑制作用。
Canine interferon alpha subtype 1（CaIFN-α1）mature protein encoded gene was sub-cloned from pMD18-T-CaIFN-α which contain complete gene of CaIFN-α1.The sub-cloned gene was inserted into pPICZα-A vector and resulted in pPICZα-A-CaIFN-α1.The recombinant plasmid was linearized with Sac Ⅰ and was then transformed into Pichia pastoris X-33 strain by electroporation.The existing of CaIFN-α1 gene in the transformed yeast cells was detected by PCR using CaIFN-α1 primers.Whether the CaIFN-α1 gene was integrated into the alcohol oxidase promotor（AOX1）locus on yeast chromosome was verified by amplification with AOX1 primers.The identified transformants were isolated and assayed for the expression of CaIFN-α1.The expression of CaIFN-α1 was induced with methanol.The existing of CaIFN-α1 in the supernatant of culture was firstly detected through SDS-PAGE,and then the positive samples were further identified by antiviral activity testing.There was a protein with relative molecular weight of approximately 2.7×104 in the supernatant.In contrast with the deduced theoretical molecular weight of CaIFN-α1,the expressed protein was larger which may be due to the difference of glycosylation.The content of the secreted CaIFN-α1 was about 96 mg·L-1 and its antiviral activity was about 1.45×106 U·mL-1.The specific activity of porcine interferon beta produced by the Pichia pastoris was approximately 1.49×107 U·mg-1.Bioactivity of CaIFN-α1 was highly species-specific,which showed high antiviral activity in canine cell MDCK and low antiviral activity in CEF（chicken）,but not in the F81（feline）and MDBK（bovine）.The antiviral activity of recombinant CaIFN-α1 aganist canine distemper virus（CDV）and pseudorabies virus（PrV）was also assessed in the present experiment.And the result indicated that CaIFN-α1 could effectively inhibit the replication of CDV and PrV in MDCK cells.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2008年第4期107-112,共6页
Journal of Nanjing Agricultural University
基金
国家青年自然科学基金项目(30600018)
南京农业大学青年科技创新基金项目(KJ05013)
关键词
犬α干扰素
毕赤酵母
分泌表达
抗病毒活性
canine interferon alpha subtype 1
Pichia pastoris
secreted expression
antiviral activity
