摘要
干扰素在病毒性疾病的防治上,具有很高的临床应用前景。为了林麝干扰素的研究与应用,使用同源克隆方法首次克隆得到9条林麝干扰素α基因序列,序列全长均为570 bp,编码189个氨基酸,前23个氨基酸为信号肽,具有4个保守的半胱氨酸残基和5个保守的脯氨酸残基。9种亚型之间,核酸序列同源性为97.0%-99.6%,氨基酸序列同源性为93.7%-99.5%。蛋白结构主要由5个α-螺旋和无规则卷曲构成,三级结构与牛和人干扰素α高度相似。结构域预测包含干扰素受体IFNAR结合位点。首次构建了林麝干扰素α表达质粒并成功使用毕赤酵母表达出林麝干扰素α,确定了1%的最佳甲醇诱导浓度与96 h的最佳诱导时间。使用qPCR首次确定了林麝干扰素α对林麝肺成纤维细胞FMD-C1干扰素刺激基因具有转录调控作用,并具有剂量依赖性,调控作用在6 h达到最强。CCK-8法确定了林麝干扰素α可以抑制FMD-C1细胞的增殖,抗增殖作用也具有剂量依赖性。以上结果为林麝干扰素α的抗病毒研究与临床应用提供了理论基础。
Interferon(IFN)has a high clinical application prospect in the prevention and treatment of viral diseases.In order to study and apply the IFN-α of forest musk deer(FMD),nine IFN-α gene sequences of FMD were cloned for the first time via homologous cloning method.Bioinformatics analysis showed that all the sequences were 570 bp and encoding 189 amino acids.Amino acid 1 to 23 were signal peptides.All of them had 4 conserved cysteine residues and 5 conserved proline residues.Among the 9 subtypes,the nucleotide sequence homology was 97.0%-99.6%,and the amino acid sequence homology was 93.7%-99.5%.The secondary structure was mainly composed of 5 α-helices and random coil.The tertiary structure was highly similar to bovine and human IFN-α,and domain predicted that it contained interferon receptor IFNAR binding sites.The expression plasmid of FMD IFN-α was constructed for the first time,and the IFN-α was successfully expressed by Pichia pastoris.The optimal methanol concentration of 1% and the optimal induction time of 96 h were determined.qPCR was used to confirm that IFN-α of FMD regulated the transcription of interferon-stimulated gene in lung fibroblasts FMD-C1 in a dose-dependent manner,and the regulatory effect reached the maximum at 6 h.CCK-8 assay confirmed that FMD IFN-α inhibited the proliferation of FMD-C1 cells in a dose-dependent manner.These results provide a theoretical basis for the antiviral research and application of IFN-α from FMD.
作者
杨威
伍茜
程建国
罗燕
王印
杨泽晓
姚学萍
YANG Wei;WU Xi;CHENG Jian-guo;LUO Yan;WANG Yin;YANG Ze-xiao;YAO Xue-ping(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130;Sichuan Institute of Musk Deer Breeding,Chengdu 611800)
出处
《生物技术通报》
CAS
CSCD
北大核心
2022年第1期194-204,共11页
Biotechnology Bulletin
基金
四川省省级科研院所科技成果转化项目(2020JDZH0024)。
关键词
林麝
干扰素
毕赤酵母
转录调控
forest musk deer
interferon
Pichia pastoris
transcriptional regulation
