摘要
为表达具有抗病毒活性的梅花鹿干扰素β1(IFN-β1),本研究通过PCR扩增梅花鹿IFN-β1基因,将其克隆至原核表达载体p ET-28a中,构建重组质粒p ET-IFNβ1,并将其转化至大肠杆菌Rosetta(DE3)感受态细胞中,经IPTG诱导后,得到高效表达。SDS-PAGE与western blot分析结果表明,梅花鹿IFN-β1重组蛋白的分子量大小约为24 ku,表达产物主要为不溶性的包涵体,表达量占菌体总蛋白的59.02%。将Ni柱纯化的p ET-IFNβ1诱导表达产物复性后,细胞病变抑制法检测表明表达产物具有抗病毒活性,其抗病毒活性可以达到10×53.81U/0.1 m L。梅花鹿INF-β1的表达为梅花鹿INF-β1的应用奠定了基础。
To express the sika deer interferon batal （IFN-β1）, the IFN-β1 gene was amplified by PCR from genome DNA extracted from liver of sika deer, cloned and sequenced. The results showed the sequence of sika deer IFN-β1 gene was 498 bp, which encodes 165 amino acid residues. Then, the IFN-β1 gene was inserted into pET-28a vector to construct recombinant plasmid of pET-IFNβ1, pET-IFNβ1 was transformed into E.coli Rosseta （DE3）. The recombinant IFN-β1 （rIFN-β1） was expressed by IPTG inducing. The expressed rIFN-β1 was about 24 ku and mainly in the form of inclusion body which was account for 59.02% of the total bacteria protein. The rIFN-β1 was further purified by the Ni column and renatured. Then, cytopathic inhibition method detected that rIFN-β1 possess antiviral activity which was about 10×5^3.82 U/0.1 mL. This study provided a basis for further study and application of sika deer IFN-β1.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2015年第2期136-139,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
吉林省自然科学基金(201115194)
国家国际科技合作专项(2011DFA32900)
关键词
梅花鹿
IFN-β1基因
原核表达
活性鉴定
sika deer
IFN-β1 gene
prokaryotic expression
activity identification
