摘要
目的:构建干扰素β1a(IFN-β1a)的CHO表达体系,探讨人IFN-β1a在真核细胞中的表达效果。方法:采用全基因合成法获取重组人IFN-β1a基因,并通过点突变将IFN-β1a的17位半胱氨酸进行修饰,合成的基因经PCR扩增,连接入pSV2-dhfr质粒中,构建重组真核表达质粒pSV2-dhfr-IFN-β1a。将质粒转染至CHO-dhfr-细胞中,经MTX加压筛选,获得稳定生长的能够持续表达IFN-β1a的单克隆细胞株;提取细胞基因组DNA,进行PCR鉴定。收集细胞培养上清液,采用Wish细胞病变抑制法检测转染细胞中IFN-β1a的抗病毒活性。结果:重组真核表达质粒pSV2-dhfr-IFN-β1a经PCR及双酶切鉴定证明构建正确;以转染细胞基因组DNA为模板,可扩增出IFN-β1a基因;转染后重组细胞表达的IFN-β1a具有抗病毒活性,达到3×105 IU.mL-1。结论:IFN-β1a基因真核表达质粒构建成功,并在CHO细胞中成功表达了具有较高生物学活性的IFN-β1a蛋白。
Objective To construct the CHO expression system of interferon-β1a（IFN-β1a） gene and to explore the expression efficacy of human IFN-β1a gene in eukaryotic cells.Methods The synthesized IFN-β1a gene was amplified and cloned into plasmid pSV2-dhfr.Cysteine 17 was mutated to serine.The constructed recombinant plasmid pSV2-dhfr-IFN-β1a was transfected to CHO-dhfr-cells,and the monoclonal cell strains growing stably were obtained by MTX pressure screening.The genomic DNA of transfected cells was extracted;target gene was identified by PCR.The antiviral activity of IFN-β1a in transfected cells was determined by Wish cytopathic inhibition test.Results Both PCR and restriction enzyme analysis showed that the recombinant plasmid pSV2-dhfr-IFN-β1a was constructed correctly.The IFN-β1a gene was amplified by using the genomic DNA of transfected cells as template.The expressed IFN-β1a showed high antiviral activity up to 3×105 IU·mL-1.Conclusion A eukaryotic expression vector of IFN-β1a gene is successfully constructed,and the IFN-β1a protein with biological activity is expressed in CHO cells.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2013年第2期204-208,共5页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金资助课题(21072075
20772046)
作者简介
王妍（1962-），女，吉林省长春市人，研究员，主要从事细胞培养技术、生物制品工艺和生物制品综合技能等方面的研究。
[通信作者]解桂秋（Tel：0431—85155212，E-mail：jiegq@jtu．edu．cn）
