Expression of function-unknown gene C16orf68 in different hepatic cell lines

Objective To establish the prokaryotic expression vector of human gene C16orf68,and get the C16orf68 recombinant protein.To prepare the C16orf68 specific rabbit polyclonal antibody,and observe the expression of C16orf68 in different cell lines.Methods C16orf68 cDNA was gotten from LX2 cells and coloned into the prokaryotic expressive vector pET-32a(+),and the recombinant plasmid was transformed into E.coil BL21.The expression of C16orf68 protein was induced by IPTG and analyzed by SDS-PAGE and Western blot.The recombinant protein was also confirmed with mass spectrometry.Then the pET-32a(+)-C16orf68 recombinant protein was used to immunize New Zealand rabbits to obtain polyclonal antibody.The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA.The expression of C16orf68 in different cell lines was observed by Western blot.Results The C16orf68 recombinant protein was highly expressed.The recombinant C16orf68 had same DNA sequence with GenBank and had the same molecular weight with prediction accessed by Western blot.The protein sequence was confirmed by Mass spectrometry.ELISA analysis indicated the titer of polyclonal antibody1∶ 320 000.The high specificity was confirmed by Western blot.The expression of C16orf68 in HepG2 cells was higher than that in LX2 cells,but lower than that in HepG2.2.15 cells.Conclusion The recombinant C16orf68 fusion protein was correctly constructed and the specific antibody was obtained,which could provide valuable tools for the investigation on the biological function of C16orf68.HBV replication may up-regulate the expression of C16orf68 in hepatocytes.