Effect of Kozak Sequnce +4G on the Expression of Human Amyloid Precursor Protein 751 Fused with EGFP in CHO Cells

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.