摘要
通过PCR技术从罗曼鸡肝脏基因组中扩增鸡α干扰素(ChIFN-α)全基因,并克隆和测序。序列分析表明,ChIFN-α基因全长为582 bp,亚克隆其成熟蛋白编码基因489 bp,利用基因重组技术构建了重组质粒,使ChIFN-α置于原核表达载体pQE30的T5启动子下并同6×His(多聚组氨酸标签)-Tag融合。经酶切和PCR鉴定,DNA测序证实重组质粒pQEChIFN-α构建正确;将pQEChIFN-α转化大肠杆菌M15感受态细胞,用IPTG诱导表达。SDS-PAGE和Westernblot证实表达出分子质量为19.80 kDa的融合蛋白。表达的蛋白以包涵体形式存在,经变性、复性处理后,在Vero细胞上抗水泡性口炎病毒的活性为1.16×103U/mg。本研究成功表达了ChIFN-α,表达的重组蛋白具有一定的生物活性。
The full length of chicken interferon alpha（ ChIFN-α ）gene was amplified by polymerase chain reaction （PCR）from the genome DNA of Luoman chicken liver,and then sequenced. The amplified gene fragment was 582 bp,of which the 489 bp coding region was subeloned into prokaryotie expression vector pQE30. The recombinant plasmid pQEChIFN-α was identified by PCR, enzyme digestion and DNA sequencing. SDS-PAGE and Westem blot showed that a 19.80 kDa fusion protein was expressed in the form of inclusion body with good immunity. After being denatured and renatured, the activity of inclusion body was detected by inhibiting the cytopathic effect. The activity of recombinant ChIFN-α against vesicular stomatitis virus at Vero cell is 1.16 × 10^3 U/mg.
出处
《华北农学报》
CSCD
北大核心
2009年第1期40-43,共4页
Acta Agriculturae Boreali-Sinica
基金
国家"十一五"科技支撑计划专项(2006BAD06A08)
关键词
鸡
Α干扰素
克隆
原核表达
抗病毒活性
Chicken
IFN-α
Clone
Prokaryotic expression
Antiviral activity
