[1]王永娟,郭艳丽,吴植,等.重组鸭α-干扰素的抗病毒活性[J].江苏农业学报,2019,(06):1402-1406.[doi:doi:10.3969/j.issn.1000-4440.2019.06.019]
　WANG Yong-juan,GUO Yan-li,WU Zhi,et al.Antiviral activity of recombinant duck α-interferon[J].,2019,(06):1402-1406.[doi:doi:10.3969/j.issn.1000-4440.2019.06.019]
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重组鸭α-干扰素的抗病毒活性()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2019年06期
页码:
1402-1406
栏目:
畜牧兽医·水产养殖
出版日期:
2019-12-31

文章信息/Info

Title:
Antiviral activity of recombinant duck α-interferon
作者:
王永娟1; 郭艳丽1; 2; 吴植1; 王安平1; 郭长明1; 吴双1; 洪伟鸣1; 秦枫1; 朱善元1
（1.江苏农牧科技职业学院/江苏省兽用生物制药高技术研究重点实验室，江苏泰州225300； 2. 南京师范大学生命科学学院，江苏南京210000）
Author(s):
WANG Yong-juan1; GUO Yan-li1; 2; WU Zhi1; WANG An-ping1; GUO Chang-ming1; WU Shuang1; HONG Wei-ming1; QIN Feng1;  ZHU Shan-yuan1
(1.Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Provincial Key Laboratory of Veterinary Bio-pharmaceutical High Tech Research, Taizhou 225300, China; 2.College of Life Sciences, Nanjing Normal University, Nanjing 210000, China)
关键词:
鸭; α-干扰素; 原核表达; 纯化; 抗病毒活性
Keywords:
duck;  α-interferon; prokaryotic expression; purification; antiviral activity
分类号:
S858.32;S859.7
DOI:
doi:10.3969/j.issn.1000-4440.2019.06.019
文献标志码:
A
摘要:
本研究参考大肠杆菌密码子的偏好性，对GenBank中已经发表的鸭α-干扰素基因序列进行优化，人工合成后与原核表达载体pET30a-ELP连接，构建原核表达质粒pET30a-DuIFNα-ELP。将重组表达质粒转化至大肠杆菌BL21 (DE3) 中，IPTG诱导表达重组蛋白DuIFNα-ELP。根据类弹性蛋白多肽 (ELP) 具有温度敏感的可逆相变特性，通过重复可逆相变循环(ITC)纯化重组蛋白质；纯化的蛋白质主要以包涵体的形式存在，通过变性、复性处理后，采用细胞病变抑制法分别在MDCK/VSV和DEF/VSV中检测重组蛋白质的抗病毒活性。结果表明，合成的重组鸭α-干扰素基因能成功表达，重组蛋白DuIFNα-ELP分子质量约80 000，纯化后的重组蛋白质纯度约90%。通过抗病毒试验检测重组DuIFNα-ELP在MDCK/VSV系统中的抗病毒活性为1.0×106U/ml，比活性为1.25×106 U/mg；在DEF/VSV系统中的抗病毒活性为1.0×107 U/ml，比活性为1.25×107 U/mg，比MDCK/VSV系统中活性高10个单位, 验证了干扰素的抗病毒活性与受体细胞的应答可能存在一定关系。这一研究结果为鸭α-干扰素防治禽类病毒性疾病的探索奠定了基础。
Abstract:
With reference to the codon preference of Escherichia coli, the duck α-interferon gene sequence published in GenBank, after artificial synthesis, it was ligated with prokaryotic expression vector pET30a-ELP to construct prokaryotic expression plasmid pET30a-DuIFNα-ELP. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3), induced by IPTG for DuIFNα-ELP expression. According to the temperature-sensitive reversible phase transition property of elastin-like polypeptide (ELP), the recombinant protein was purified by repeated inverse transition cycling (ITC). The purified protein mainly existed in the form of inclusion bodies. After denaturation and renaturation treatment, the antiviral activity of the recombinant protein was detected in MDCK/VSV and DEF/VSV by micro-cytopathic inhibition method. The results showed that the recombinant α-interferon gene could be successfully expressed. The molecular weight of recombinant protein DuIFNα-ELP was about 80 000, and the purity of the purified recombinant protein was about 90%. Antiviral assay showed that the antiviral activity of recombinant DuIFNα-ELP in MDCK/VSV system was 1.0×106 U/ml, the specific activity was 1.25×106 U/mg. The antiviral activity in DEF/VSV system was 1.0×107 U/ml, specific activity was 1.25×107 U/mg, which was 10 units higher than the activity in the MDCK/VSV system. It is verified that the antiviral activity of interferon may be related to the response of recipient cells. These results lay the foundation for the exploration of duck α-interferon in the clinical prevention and treatment of avian viral diseases.

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备注/Memo

备注/Memo:
收稿日期：2019-04-10 基金项目：江苏省高等学校自然科学研究重大项目（19KJA520008）；江苏农牧科技职业学院横向配套课题（NSFPT201710）；2017年省高校协同创新中心立项课题（NSFKF201705）；江苏省农业科技自主创新基金项目[CX（18）1004] 作者简介：王永娟（1980-），女，江苏海门人，博士，教授，主要从事动物传染病与生物制药研究。（E-mail）43088591@qq.com。郭艳丽为共同第一作者。 通讯作者：朱善元，（E-mail）jstzzsy126.com
更新日期/Last Update: 2020-01-09