生物技术通报 ›› 2022, Vol. 38 ›› Issue (1): 187-193.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0195

• 研究报告 • 上一篇    下一篇

羊α干扰素在家蚕中的表达及抗小反刍兽疫病毒活性测定

李丹1(), 杜梦潭1, 修明霞2, 刘兴健1, 张志芳1, 李轶女1()   

  1. 1.中国农业科学院生物技术研究所,北京 100081
    2.乳山市果茶蚕工作站,烟台 264500
  • 收稿日期:2021-02-21 出版日期:2022-01-26 发布日期:2022-02-22
  • 作者简介:李丹,女,硕士研究生,研究方向:病毒微生物学;E-mail: lidancaas@163.com
  • 基金资助:
    国家重点研发计划(2016YFD0500108);国家自然科学基金项目(32072796);国家自然科学基金项目(32002236)

Expression of Sheep Interferon Alpha in Silkworm and Determination of Its Activity Against Peste Des Petits Ruminants Virus

LI Dan1(), DU Meng-tan1, XIU Ming-xia2, LIU Xing-jian1, ZHANG Zhi-fang1, LI Yi-nv1()   

  1. 1. Institute of Biotechnology,Chinese Academy of Agricultural Sciences,Beijing 100081
    2. Rushan City Fruit Tea Silkworm Workstation,Yantai 264500
  • Received:2021-02-21 Published:2022-01-26 Online:2022-02-22

摘要:

干扰素具有广谱抗病毒、抗肿瘤活性,可增强机体抗病毒能力,已经广泛应用于病毒性疾病的防控。小反刍兽疫病毒是危害山羊、绵羊等小反刍动物常见的病毒之一,该病毒的传播对全球养殖业造成了严重的影响。为了表达具有高效抗病毒活性的羊α干扰素(OviIFN-α),将OviIFN-α基因序列根据家蚕密码子偏好性进行优化合成,构建pVL1393-OviIFN-α转移载体,将转移载体与ORF1629缺失型杆状病毒基因组DNA利用脂质体介导的转染法在BmN细胞中进行重组,重组病毒感染家蚕,在蚕血淋巴液中获取重组蛋白OviIFN-α。利用微量细胞病变抑制法在BHK-VSV*GFP系统中检测到该蛋白表达量可达6.5×105U/mL左右。小反刍兽疫病毒增殖抑制实验显示,羊α干扰素对小反刍兽疫病毒在BHK细胞中的复制产生了明显的抑制作用,IC50为136.93 U左右。该结果为羊α干扰素用于小反刍兽疫疫情防控提供了重要的理论依据。

关键词: 羊α干扰素, 杆状病毒表达系统, 小反刍兽疫病毒, 抗病毒活性

Abstract:

Interferon has broad-spectrum anti-viral and anti-tumor activities and can enhance the body’s anti-viral ability,thus interferon has been widely used in the prevention and control of viral diseases. Peste des petits ruminants virus(PPRV)is one of the common viruses that harm small ruminants such as goats and sheep. The spread of this virus has resulted in a serious impact on the global breeding industry. In order to express sheep interferon alpha(OviIFN-α)with high antiviral activity,the OviIFN-α gene sequence was optimized and synthesized according to the codon preference of the silkworm to construct pVL1393-OviIFN-α transfer vector,and then the transfer vector and baculovirus genomic DNA with deleted of ORF1629 sequence was recombined in BmN cells by liposome-mediated transfection. The recombinant virus infected the silkworm,and the recombinant protein OviIFN-α was obtained from the silkworm hemolymph. The antiviral titer of the protein detected in the BHK-VSV*GFP system using the cytopathic inhibition method reached about 6.5×105 U/mL. In addition,the experiments on the proliferation inhibition of PPRV showed that OviIFN-α significantly inhibited the replication of PPRV in the BHK cells,with an IC50 of about 136.93 U. This result provided an important theoretical basis for OviIFN-α to be used in the prevention and control of peste des petits ruminants disease.

Key words: sheep interferon alpha, baculovirus expression system, peste des petits ruminants virus, antiviral activity