Objective To construct the GFP-tagged eukaryotie expression vector of NLRC5 and observe its expres-sion in mouse macrophage RAW264 . 7 . Methods The cDNA of NLRC5 was obtained from mouse macrophage RAW264. 7 cells, amplified by PCR and cut with double enzyme EcoR I and BamHI, then inserted into the eu-karyotic expression vetor pEGFP-C2. The recombinant vector was verified by PCR, restriction enzymes cut and se-quencing identified. Then transfected into mouse macrophage RAW264. 7 cells and the expression of pEGFP-C2-NLRC5 was monitored by fluorescence, PCR and Western blot. Results To deal with recombinant pEGFPC2-NL-RC5 with double digestion, then fragments of NLRC5 could be seen, also GFP could be detected in the transfected RAW264. 7 cells. NLRC5 gene expression could be detected by PCR,and its protein expression was detected by Western blot. Conclusion Eukaryotic expression vector of NLRC5 is successfully constructed, and the fusion ex-pression of NLRC5 protein and GFP can be detected in RAW264 . 7 .%目的构建鼠源pEGFP-C2-NLRC5表达质粒,并观察其在 RAW264.7细胞中的表达。方法从小鼠巨噬细胞RAW264.7中获得 NLRC5基因的 cDNA,用 EcoR I和BamH I限制性内切酶双酶切后连接至pEGFP-C2载体上。表达载体经PCR、限制性内切酶酶切和DNA序列分析正确后转染至小鼠巨噬细胞RAW264.7中,荧光显微镜下观察绿色荧光蛋白表达, RT-PCR法鉴定其转录, Western blot法分析其蛋白表达。结果进行双酶切鉴定该质粒可见NLRC5 DNA片段,转染重组质粒后可观察到绿色荧光蛋白的表达, RT-PCR法可检测到NLRC5基因的转录, Western blot法可检测到NLRC5蛋白表达。结论成功构建重组pEGFP-C2-NLRC5表达载体, NLRC5蛋白可与绿色荧光蛋白在RAW264.7细胞中融合表达。
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