脑红蛋白重组腺病毒表达载体的构建与鉴定

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BJ5183细菌，制备BJ5183-pAdEasy-l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Western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。

Construction and identification of recombinant adenoviruses expressing neuroglobin

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 neuroglobin(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 pShuttle-CMV to form pShuttle-CMV-NGB.Electrical transformation of the plasmid pAdEasy-1 into E.coli BJ5183 strain was performed to prepare BI5183-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.