摘要
血凝素(hemagglutinin,HA)是禽流感病毒的主要抗原之一,原核表达HA是该抗原特性研究和疫苗制备的关键。利用分子生物学方法构建H5N1亚型的禽流感病毒HA基因原核表达载体,转化至大肠埃希菌Rosetta菌株,用不同IPTG浓度和时间诱导HA表达,检测蛋白表达量,分析不同诱导条件对HA表达效率的影响,以确定最佳诱导条件。重组表达载体pET32a-HA经BamHⅠ和EcoRⅠ双酶切鉴定,获得预期大小的DNA片段,说明HA基因成功插入质粒pET32a。Western blot检测结果表明,IPTG诱导重组菌株成功表达HA蛋白,当诱导浓度为0.1mmol/L,诱导时间为6h时,HA蛋白表达量最高。研究结果为HA抗原制备及禽流感疫苗的研究奠定基础。
Hemagglutinin(HA)is one of vital antigens of avian influenza virus.Expression of HA in prokaryotic cells plays an important role in exploration of HA characteristics and preparation of vaccine.In this study,HA of H5N1 was cloned into the prokaryotic expression vector,and transformed into Escherichia coli Rosetta.In order to optimize induction conditions,recombinant bacteria were induced with different IPTG concentrations and induction time,and then ratio of target protein/total proteins was measured.The result of Bam HⅠand Eco RⅠdigestion of pET32a-HA demonstrated that the expected length of HA fragment was inserted into pET32a.Western blot result illustrated that HA protein was successfully expressed in the recombinant strain via IPTG induction.With optimal conditon of 0.1 mmol/L IPTG and 6 h induction time,HA expression level reached highest.This study provided the foundation for the preparation of HA antigen and avian influenza vaccine research.
作者
杨理凯
郭苗苗
杜伟立
丁锐
王令
张涛
路宏朝
YANG Li-kai;GUO Miao-miao;DU Wei-li;DING Rui;WANG Ling;ZHANG Tao;LU Hong-zhao(School of Biological Science and Engineering,Shaanxi University of Technology,Hanzhong,Shaanxi,723001,China)
出处
《动物医学进展》
北大核心
2018年第8期10-14,共5页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31402071)
陕西理工大学科研计划资助项目(SLGQD14-02)
陕西理工大学大学生创新创业训练计划项目(UIRP15020)
关键词
禽流感病毒
血凝素
原核表达
诱导条件
Avian influenza virus
hemagglutinin
prokaryotic expression
induction condition