小鼠HSP27基因腺病毒表达载体的构建与鉴定
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国家自然科学基金资助项目(30800394,30801236),江苏省科教兴卫工程基金资助项目(LJ200606)


Construction and identification of recombinant adenovirus vector carrying mouse HSP27
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    摘要:

    目的:为深入研究热休克蛋白27(heat shock protein 27,HSP27)的生理与病理功能,应用PAdEasy腺病毒载体系统构建带荧光蛋白的小鼠HSP27重组腺病毒,并进行鉴定-方法:采用基因技术,将目的基因克隆至穿梭质粒pAdTrack-CMV中,通过PCR-序列测定-酶切鉴定;利用PAdEasy系统进行重组,在293细胞中包装和扩增,然后体外感染小鼠精原母细胞,Western blot检测HSP27蛋白表达-结果:测序和酶切鉴定重组腺病毒质粒构建正确;经293细胞包装后可观察到绿色荧光,收获高滴度重组腺病毒体外感染小鼠精原母细胞,并且可正确表达HSP27-结论:应用PAdEasy系统重组技术成功构建了小鼠HSP27带荧光蛋白腺病毒载体,并在小鼠精母细胞系进行体外有效表达,为进一步研究HSP27的功能奠定基础-

    Abstract:

    Objective:To investigate the physiological and pathological function of HSP27 by construct and identify the recombinant adenoviruses containing green fluorescence protein(GFP)and mouse HSP27 gene with PAdEasy system. Methods:The coding region of HSP27 was subcloned into the shuttle vector pAdTrack-CMV to form pAdTrack-CMV-HSP27. The identification was performed by PCR,sequencing and restriction digest. Chemical transformation of the plasmid pAdEasy-1 into E. coli BJ5183 strain was performed to prepare BJ5183-pAdEasy-1 as the competent bacterium,in which pAdEasy-1 and pAdTrack-CMV-HSP27 were cotransformed. Finally,the recombinant adenovirus containing the coding region of HSP27 gene was constructed by transfecting 293 cells with linearized adenoviral genomes of Ad-CMV-HSP27,and produce was used to infect mice spermatocyte,the expression of HSP27 protein was detected by Western blot. Results:The recombinant plasmids identified by PCR and directly sequencing were positive. GFP was observed after construction,high concentration of spermatocytes infected by recombinant adenortrus was harvested,and HSP27 protein expressed correctly. Conclusion:The recombinant adenoviruses expressing HSP27 and GFP using AdEasy system,which will facilitate further functional studies of HSP27,are successfully performed in this study.

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刘金娟,马 翔,刁飞扬,李 梅,薛 凯,崔毓桂,刘嘉茵.小鼠HSP27基因腺病毒表达载体的构建与鉴定[J].南京医科大学学报(自然科学版),2009,29(7):944-948

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  • 收稿日期:2009-02-04
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