Abstract：Objective: To construct the secretory expression vector containing human immunodeficiency virus type I (HIV-1) negative regulation factor (Nef) and enhanced green fluorescent protein (EGFP) gene, and appraise the expression of Nef-EGFP fusion protein in the eukaryotic cell and the secretion into the supernatant. Methods: The Nef and EGFP fragment was amplified by PCR, then cloned into the secretory expression vector pSecTag2B. The recombinant vector pSecTag2B-Nef-EGFP was transfected into the 293T cells, observed the expression of GFP, then harvested the culture media and cells, the fusion protein NefEGFP was confirmed by Western blot and ELISA. Results: The recombinant secretory expression vector carried the Nef and EGFP gene was successfully constructed. Western blot analysis revealed that the Nef-EGFP fusion protein can be correctly transcripted and translated in 293T cells, ELISA analysis discovered it can be secreted into the supernatant, the level was 1.7 ng/ml. Conclusion: The recombinant secretory expression vector pSecTag2B-Nef-EGFP was constructed successfully, Nef-EGFP fusion protein could be expressed in 293T cells, and secreted to the supernatant.
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2012, Vol. 22 
