铁蛋白与犬α干扰素的融合表达及抗病毒活性检测
Prokaryotic Expression and Antiviral Activity of Canine Interferon Fused with Fe Protein
宋若楠 1刘春草 2李航 2王真真 1贾楠楠 2朱杰 2李传峰 2程松 3梁留存 3刘光清 2王金泉 4孟春春1
作者信息
- 1. 新疆农业大学,乌鲁木齐 830000;中国农业科学院上海兽医研究所,上海 200241
- 2. 中国农业科学院上海兽医研究所,上海 200241
- 3. 德国纳博科临中国检测实验室,上海 200241
- 4. 新疆农业大学,乌鲁木齐 830000
- 折叠
摘要
为获得治疗犬病毒性传染病的特效药物,本研究将铁蛋白与犬α干扰素进行融合表达.首先根据GenBank中公布的犬α干扰素序列(IFN2a)合成模板,通过融合PCR连入铁蛋白编码基因的上游,并将该重组基因克隆至原核表达载体pCold-TF中,获得重组质粒pCold-TF-CaIFN2a-Fe.转化BL21后用异丙基-β-D硫代半乳糖苷(IPTG)诱导,经聚丙烯酰胺凝胶电泳(SDS-PAGE),蛋白免疫印迹(Western blot)鉴定,获得约89 kDa的可溶性重组目的蛋白.重组蛋白经镍柱纯化后,使用犬肾细胞/水疱疹性口炎病毒(MDCK/VSV)微量细胞病变抑制法检测抗病毒活性,效价确定为8×104 IU/mg,显示出良好的应用前景.
Abstract
In this study,ferritin was fused with canine interferon α to obtain specific drugs for canine viral infectious diseases.Firstly,according to the synthesis template of canine interferon α sequence(IFN2a)published in GenBank,the ferritin encoding gene was linked to the upstream by fusion PCR and the recombinant gene was cloned into the prokaryotic expression vector pCold-TF to obtain the recombinant plasmid pCold-TF-CaIFN2a-Fe.After transformation into BL21,the recombinant protein was induced by IPTG and identified by SDS-PAGE and Western blot.The soluble recombinant protein of 89 kDa was obtained and purified with nickel column.Its antiviral activity was detected using MDCK/VSV microcytopathy inhibition assay.The antiviral titer was 8×104 IU/mg,showing a good application prospect.
关键词
铁蛋白/犬α干扰素/融合表达/抗病毒活性Key words
Ferritin/canine interferon α/fusion expression/antiviral activity引用本文复制引用
基金项目
中国农业科学院青年英才培育工程专项经费()
出版年
2024
国家科技期刊平台
NSTL
万方数据