摘要
验证通过分子信息理论预测获得的高频干扰素(HFINF)抗病毒活性。以软件InsightⅡ和CE预测获得高频干扰素基因序列,通过分子克隆技术,将高频干扰素基因克隆到pGEX-4T-2载体中,构建高效表达质粒pGEX-4T-2-HFIFN,转化至大肠杆菌E.coliBL21(DE3)中,构建高效表达菌株,通过优化培养条件,逐级扩大培养、收集菌体,裂解菌体,将表达产物通过GST柱纯化,经SDS-PAGE鉴定,获得高纯度的HFIFN。结果表明,在菌体密度OD约为0.6时,加入体积浓度为1 mmol/L的IPTG,25℃下诱导4 h可获得含量最高的可溶性表达,将纯化的高频干扰素,通过HA细胞培养,加入VSV病毒验证实验,鉴定重组高频干扰素抗病毒活性。结果表明重组高频干扰素具有抗病毒活性,分子信息理论和实践是吻合的,这为进一步研究奠定了基础。
In order to identify the activity of anti-virus of high-frequency interferon（HFIFN）,which was predicted by bioinformatics,HFIFN gene sequence was predicted and gained by Insight Ⅱ,CE and some other related internet.PGEX-4T-2-HFIFN was constructed by a series of molecular cloning techniques,and converted to E.coli BL21（DE3） successfully.After the cells were cultured on large scale,collected and lysised,the expression products were purified by GST column,and identified by SDS-PAGE.When value of density OD of E.coli BL21（DE3） is up to 0.6,amplification culture time,IPTG concentration,induction temperature and induction time were 1mmol/L,25℃,4h,respectively,and the highest value of expressed HFINF appeared.The antivirus activity of HFIFN was detected by cell culture of HA and VSV in vitro.The results proved that the purified recombinant HFIFN has a high activity against VSV.All the above indicated that the molecular information theory was useful in predicting HFINF activity,and the HFIFN can be used for the other research.This provided a basis for the further research.
出处
《药物生物技术》
CAS
CSCD
2011年第1期6-10,共5页
Pharmaceutical Biotechnology
基金
安徽省教育厅自然科学基金(2006KJ381B)
关键词
重组高频干扰素
pGEX-4T-2
纯化
活性鉴定
Recombinant high-frequency interferon
pGEX-4T-2-HFIFN
Purification
Activity identification
