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A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
Published on: July 31, 2012
[Construction of the eukaryotic expression vector of bioactive fragment of human bactericidal/permeability-increasing
Jia-yun Liu1, Yin Long, Yue-yun Ma
1Research Center for Clinical Molecular Biology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. jiayun@fmmu.edu.cn
Aim:
To construct the eukaryotic expression vector of the cDNA sequence encoding bioactive N-terminal fragment of human bactericidal/permeability-increasing protein (BPI) and express it in CHO cells.
Methods:
Total RNA was extracted from human polymorphonuclear leukocytes (PMN) and subjected to reverse transcription, then the human BPI cDNA gene was amplified by nested PCR. The PCR product was cloned into pUC19 plasmid and confirmed by restriction enzyme digestion and DNA sequencing. Then the specific BPI encoding fragment was subcloned into pcDNA3 plasmid to form pcDNA-BPI(N) eukaryotic expression vector. CHO cells were transfected with the recombinant plasmid and the stable clones were selected by G418. The expression of BPI was identified by immunofluorescent assay.
Results:
Restriction enzyme digestion and DNA sequence analysis revealed that the sequence encoding signal peptide and bioactive N-terminal fragment of BPI had six nucleotide substitutions, compared with that of the established human BPI sequence. The expression products of the selected CHO positive cell clones were detected by anti-BPI monoclonal antibody.
Conclusion:
The construction of the eukaryotic expression vector of bioactive fragment of human BPI and its successful expression in CHO cells are helpful to further study of the role of BPI.
