摘要
目的:观察Kozak序列(+4G)对稳定转染的人淀粉样前体蛋白(hAPP751)-EGFP融合真核表达载体在CHO细胞中表达的影响,为APP的水解代谢研究提供细胞模型。方法:分别将含Kozak序列(+4G)和不含Kozak序列的hAPP751全长基因片段亚克隆入pEGFP-N1表达载体,得到pEGFP-hAPP751(+4G)和pEGFP-hAPP751重组质粒,转染CHO细胞,通过G418筛选稳定转染细胞株,再用倒置荧光显微镜挑取绿色荧光强的细胞进行亚克隆,并观察融合蛋白的表达强度和细胞定位,最后用EGFP抗体通过Western印迹检测融合蛋白。结果:PCR、酶切和测序证明将含Kozak序列(+4G)和不含Kozak序列的hAPP751全长基因片段分别连入了真核表达载体pEGFP-N1中;荧光显微镜下观察pEGFP-hAPP751(+4G)稳定转染细胞的细胞膜和细胞质产生较强的绿色荧光,其中在细胞质中成不均匀颗粒状分布,Western印迹检测到相对分子质量约156 000的融合表达蛋白,与预期相符;pEGFP-hAPP751转染细胞,其绿色荧光十分微弱且在整个细胞均匀分布,Western印迹检测到相对分子质量约26 000的EGFP,但检测不到预期的hAPP751-EGFP融合表达蛋白。结论:Kozak序列(+4G)可以明显促进hAPP751的表达,获得稳定转染且高水平融合表达hAPP751-EGFP的细胞株。
Objective： To investigate the effect of Kozak sequence ＋4G on expression of human amyloid precursor protein 751（hAPP751） and enhanced green fluorescent protein（EGFP） fusion gene.Methods： Two different full length hAPP751 gene fragments,one had Kozak sequence ＋4G,while the other had no Kozak sequence,were obtained through PCR with different primers,and were inserted into EGFP expression vector,pEGFP-N1.The recombinant vector was stably transfected into CHO cells by lipsome.The expression level and location of fusion protein were examined with fluorescence microscope and Western blotting analysis.Results： The recombinant eukaryotic expression plasmid was constructed and identified by PCR,restriction endonuclease and DNA sequencing analysis.The green fluorescence of pEGFP-hAPP751（＋4G） was bright and located on cellular membrane and inhomogeneous distribution in cytosol,and the fusion protein was confirmed by Western blotting,its relative molecular weight was the expected 156 000.The green fluorescence of pEGFP-hAPP751 was very weak and homogeneous distribution in cytosol,but only the EGFP was detected by Western blotting,and its relative molecular weight is about 26 000.Conclusion： The Kozak sequence ＋4G enhancd the hAPP751 expression in CHO cells,and a stable CHO cell line expressing hAPP751-EGFP was established,which may help to further studies of hAPP751.
出处
《生物技术通讯》
CAS
2011年第2期149-153,195,共6页
Letters in Biotechnology
基金
国家自然科学基金(30500085)
湖北省自然科学基金(2009CDB336)
武汉市卫生局科研基金(WX09B13)
