AIM: To construct pEGFP-C1-Ipr1 (intracellular pathogen resistance 1) eukaryotic expression vector using green fluorescence protein as reporter gene and to express it in RAW264. 7 cells. METHODS: The pET32a ( + )-Ipr1 and pECFP-C1 vectors were digested by Kpn Ⅰ /BamH Ⅰ and purified by gel extraction. The Ipr1 genes were inserted into pEGFP-C1 vector. The RAW264.7 cells were transfected with the constructs by LipofectaminTM 2000. The EGFP-Ipr1 fused protein was visualized directly under fluorescence microscope and the expression of Ipr1 was detected by Western blot and PCR at protein and gene levels respectively. RESULTS: The fragments with size of 4700 bp and 1338 bp were obtained by enzyme digestion and a fusion protein about 77 ku was observed by Western Blot. Restriction enzyme digestion and sequence analysis showed that the recom-binant vector pEGFP-C1-Ipr1 was constructed successfully, which was stably expressed in eukaryotic cells. Ipr1 gene and protein were detected in the transfected RAW264. 7 cells and the expressed EGFP was observed under fluorescence microscope. CONCLUSION: The eukaryotic expression vector named pEGFP-C1-Ipr1 is successfully constructed and the RAW264. 7 cell clone stably expressing Ipr1 is obtained, which provides a good basis for further research on the function of Ipr1 against tuberculosis.
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万方数据
维普
