[1]李华,汉武娇,范文禄,等.猫干扰素α 基因的克隆及其表达产物的抗病毒活性分析[J].中国预防兽医学报,2021,(02):186-190.[doi::10.3969/j.issn.1008-0589.201912049]
 LI Hua,HAN Wu-jiao,FAN Wen-lu,et al.Cloning, expression and antiviral activity of feline IFN-α[J].Chinese journal of preventive veterinary medicine,2021,(02):186-190.[doi::10.3969/j.issn.1008-0589.201912049]
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猫干扰素α 基因的克隆及其表达产物的抗病毒活性分析
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2021年02
页码:
186-190
栏目:
免疫学
出版日期:
2021-03-16

文章信息/Info

Title:
Cloning, expression and antiviral activity of feline IFN-α
文章编号:
:1008-0589(2021)02-0186-05
作者:
 李华汉武娇范文禄姜艳平崔文李一经徐义刚*
 (东北农业大学动物医学学院/黑龙江省动物疾病防控技术与制剂创制重点实验室,黑龙江哈尔滨150030)
Author(s):
 LI Hua HAN Wu-jiao FAN Wen-lu JIANG Yan-ping CUI Wen LI Yi-jing XU Yi-gang*
 (Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, College of Veterinary Medicine, Northeast
Agricultural University, Harbin 150030, China)
关键词:
:猫干扰素α基因克隆可溶性表达抗病毒活性
Keywords:
: feline IFN-α gene cloning soluble expression antiviral activity
分类号:
S852.4
DOI:
:10.3969/j.issn.1008-0589.201912049
文献标志码:
A
摘要:
为制备重组猫IFN-α并检测其抗病毒活性,本研究采用水泡性口炎病毒(VSV)感染结合poly I:C刺
激实验猫后,提取猫脾淋巴细胞总RNA 并反转录为cDNA,以其为模板经RT-PCR 方法扩增获得了567 bp 的猫
IFN-α基因,测序后进行生物信息学分析。结果显示,猫IFN-α有21个潜在磷酸化位点、1个N-糖基化位点和8
个O-糖基化位点,二级结构以α-螺旋为主。将该基因经BamH I/Kpn I酶切处理后克隆至pCold-TF载体,构建重组
表达质粒pCold-α,将其转化大肠杆菌BL21感受态细胞后获得了表达猫IFN-α的重组大肠杆菌pCold-α/BL21。重组
菌经IPTG诱导后SDS-PAGE检测结果显示重组猫IFN-α蛋白获得高效表达,采用His标签蛋白纯化柱纯化后得到纯
化蛋白浓度为340 mg/L。以VSV和猫冠状病毒(FCoV)为模式病毒,采用微量细胞病变抑制法检测了重组猫IFN-α的
抗病毒效果,结果显示,其抗VSV活性为5.91×105 IU/mg,抗FCoV活性可达6.25×106 IU/mg,具有良好的抗病毒活
性。本研究为猫干扰素的开发应用奠定了物质基础。
Abstract:
To prepare recombinant protein of feline IFN-α (feIFN-α) and evaluate its antiviral activity, the gene encoding the
feIFN-α was amplified by RT-PCR with the length size of 567 base pairs from the spleen lymphocytes of cat after infection with
VAS combined with poly I:C stimulation. The bioinformatics analysis showed that the feIFN-α had twenty-one potential
phosphorylation sites, one N-glycosylation site and eight O-glycosylation sites, and its secondary structure were α-helix. Subsequently,
the gene was digested with BamH I/Kpn I and subcloned into the pCold-TF vector to construct a recombinant E. coli
pCold-α/BL21 expressing the feIFN-α. After induction by IPTG, fusion protein was highly expressed by using SDS-PAGE analysis,
and 340 mg/L of target protein (21 ku) was obtained after protein purification with a histidine (His) affinity tags. The antiviral
activity of the feIFN-α was evaluated by microcytopathic-inhibiting-assay in the cells infected with either VSV or FCoV. And the
results showed that the antiviral activities of the feIFN-α against VSV and FCoV were 5.91 × 105 IU/mg and 6.25 × 106 IU/mg,
respectively. The feIFN-α had good antiviral activity, providing a reference for the further development of the feIFN-α.

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更新日期/Last Update: 2021-03-17