脑红蛋白重组腺病毒表达载体的构建与鉴定  

Construction and identification of recombinant adenoviruses expressing neuroglobin

作　　者：刘铖[1] 高艳[1] 秦树桐[1] 文圣梅[1] 王立生[1] 张成岗[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《军事医学科学院院刊》2006年第6期517-520,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家重点基础研究发展计划("973"计划)(2006CB504100);国家自然科学基金项目(90208017;30400465;30300358)资助;北京市自然科学基金重点项目(7061004);全军"十一五"医药卫生科研基金(06MA311)

摘　　要：目的:为进一步研究脑红蛋白(NGB)的生理功能并为基因治疗缺血性脑损伤提供有效的表达载体,应用细菌内同源重组方法分别构建含NGB及绿色荧光蛋白(GFP)基因的重组腺病毒表达载体,并对其进行鉴定。方法:用电转方法将腺病毒基因组质粒pAdEasy-1导入B J5183细菌,制备B J5183-pAdEasy-1细菌,再以后者作为电感受态细菌,与线性化质粒pShuttle-CMV-NGB进行同源重组,获得重组腺病毒质粒pAdEasy-NGB,转染293细胞,制备含脑红蛋白基因的重组腺病毒。结果:通过PCR、序列测定和W estern印迹杂交鉴定,确认该重组腺病毒表达载体可正确表达脑红蛋白。结论:成功地构建了表达NGB基因的重组腺病毒载体,为深入开展脑红蛋白的功能研究以及用于缺血性脑损伤疾病的基因治疗奠定了基础。Objective：To construct the recombinant adenoviruses containing human NGB and green fluorescence protein （GFP） gene with the method of homologous recombination in bacteria respectively in order to investigate the function of neuroglohin （NGB） and to provide an appropriate gene carrier for treating cerebral ischemic diseases. Methods：The coding region of NGB was subcloned into the shuttle vector pShutfle-CMV to form pShuttle-CMV-NGB. Electrical transformation of the plasmid pAdEasy-1 into E. coil BJ5183 strain was performed to prepare B15183-pAdEasy-1 as the competent bacterium, in which pAdEasy-1 and pShuttle-CMV-NGB were cotransformed. Finally, the recombinant adenovirus containing the coding region of NGB gene was constructed by transfecting 293 cells with linearized adenoviral genomes of pAdEasy-NGB. The recombinant adenovirus containing the coding region of GFP gene was constructed similarly. Results： The positive recombinant plasmids for NGB and GFP were further identified by PCR, direct sequencing and Western blot analysis. Conclusion： The recombinant adenoviruses expressing NGB and GFP using AdEasy system are successfully constructed in the present study, which will facilitate further functional studies of NGB.

关 键 词：神经系统疾病 脑红蛋白 腺病毒科 腺病毒载体 AdEasy系统 基因表达 基因疗法 

分 类 号：Q78[生物学—分子生物学]