重组MIF腺病毒载体的构建、包装与纯化及其意义  

Construction of a recombinant adenovirus vector containing MIF and its expression in human airway smooth muscle cells

作　　者：蓝海兵[1] 罗亮[2] 齐协飞[1] 龚园其[1] 陈玉[1] 

机构地区：[1]南昌大学第二附属医院综合ICU,南昌330006 [2]中山大学第一附属医院MICU,广州510080

出　　处：《江西医药》2016年第6期516-519,共4页Jiangxi Medical Journal

基　　金：国家自然科学基金(81360005);江西省自然科学基金(20122BAB215001)

摘　　要：目的采用分子生物学技术构建人巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)基因腺病毒表达载体,检测AD-MIF腺病毒转染人气道平滑肌细胞(airway smooth muscle cell,ASMC)后蛋白表达。方法用凝胶电泳、测序、Western blotting等方法鉴定MIF质粒。用PCR技术扩增MIF基因,构建MIF-EGFP基因载体,重组到p Ad腺病毒,转染HEK293细胞后扩增病毒,经3轮扩增后病毒达到合适的滴度,Western blotting检测到腺病毒感染的气道平滑肌细胞内MIF蛋白表达明显增高。结果凝胶电泳、测序和Western blotting检测MIF基因分子量、序列以及蛋白表达正确,并且成功构建MIF腺病毒,病毒浓度达2E+9PFU/ml。结论成功构建MIF腺病毒,为MIF基因治疗支气管哮喘提供了实验基础。Objective To construct a recombinant adenovirus vector containing （Macrophage migration inhibitory factor,MIF） and identify the function of the Ad-MIF. Methods Amplify MIF gene plasmid with molecular biological technique. Identify the success of MIF gene plasmid amplification with gel electrophoresis,sequencing and Western Blot technique. Amplify MIF gene with PCR technology,then recombinate gene to adenovirus vector and prepared for adenovirus packaging. Amplify virus in HEK293 cells,After 3 cycles of amplification,the titer of adenovirus containing MIF reached an appropriate level. and The protein expressed in the infected human airway smooth muscle cells significantly increased. Results It is correct when identify the MIF gene plasmid using gel electrophoresis,sequencing and Western Blot technique.The the virus titre was 2E＋9PFU/ml. Conclusions The successful construction of the recombinant adenovirus containing MIF,which provides an experimental basis for the basic re-search of MIF gene therapy on asthma.

关 键 词：巨噬细胞移动抑制因子 腺病毒载体 气道平滑肌 支气管哮喘 

分 类 号：R346[医药卫生—基础医学]