摘要
目的构建胞内病原体抗性基因1(Ipr1)和绿色荧光蛋白(GFP)基因真核共表达穿梭质粒,并在人肺腺癌细胞A549中表达。方法采用PCR方法,分别从质粒pEGFP-C1-Ipr1和pEGFP-C1中扩增Ipr1和GFP基因,将GFP基因、分枝杆菌复制子OriM和Ipr1基因同时克隆入多启动子真核共表达载体pBudCE4.1中,构建pBud-GFP-OriM-Ipr1穿梭质粒,脂质体法转染A549细胞,荧光显微镜观察GFP的表达,免疫组化方法检测Ipr1蛋白的表达。结果酶切和测序分析表明,pBud-GFP-OriM-Ipr1真核共表达穿梭质粒构建正确。转染A549细胞后,荧光显微镜下可观察到转染细胞中有GFP表达,免疫组化法可检测到Ipr1蛋白的表达,且定位于细胞核内。结论已成功构建Ipr1和GFP基因真核共表达穿梭质粒,为进一步研究Ipr1抗结核的功能奠定了基础。
Objective To construct a shuttle plasmid for co-expression of intracellular pathogen resistance gene 1 （Iprl） and green fluorescent protein（GFP） gene in human lung cancer A549 cells. Methods Iprl and GFP genes were amplified from plasmids pEGFP-CI-Iprl and pEGFP-C1 by PCR respectively. The amplified Iprl and GFP genes as well as mycobaeterium replicon OriM were cloned into eukaryotic co-expression vector pBudCE4.1, and the constructed shuttle plasmid pBud-GFP-OriM-lprl was transfected to A549 cells in mediation of liposome. The expression of GFP was observed by fluorescent microscopy, and that of Iprl protein was determined by immnnohistochemical assay. Results Both restriction analysis and sequencing proved that shuttle plasmid pBudGFP-OriM-Iprl was constructed correctly. Expressed GFP was observed by fluorescent microscopy in transfected A549 cells. Immunohistochemical assay proved that Iprl protein was expressed in transfected A549 cells and located in cell nucleus. Conclusion A shuttle plasmid for eukaryotie co-expression of Iprl and GFP genes was successfully constructed, which laid a foundation of further study on function of Iprl against tuberculosis.
出处
《中国生物制品学杂志》
CAS
CSCD
2009年第5期464-467,共4页
Chinese Journal of Biologicals
基金
国家自然科学基金(30771922)
关键词
胞内病原体抗性基因1
绿色荧光蛋白
穿梭质粒
共表达
肺癌细胞
Intracellular pathogen resistance gene 1 （Iprl）
Green fluorescent protein （GFP）
Shuttle plasmid
Co-expression
Lung cancer cells
