Cloning, expression and identification of human ATP11A and ATP11B

Abstract

Objective To construct the expression vectors carrying human Type Ⅳ-P-type phospholipids transferases 11A or 11B (ATP11A or ATP11B), and to detect the up-regulation effect on ATP11A or ATP11B. Methods The primers were designed according to ATP11A or ATP11B sequence and cloned into EZ-TTM Cloning Vector. The primers were identified by enzyme digestion and verified by sequencing. ATP11A and ATP11B sequences were digested and directed to expression vectors pLVX-IRES-mCherry and pLVX-IRES-RFP, respectively. Then the recombinant expression plasmids with immunofluorescence protein were transfected into HEK-293T using liposomes. Finally, the expression of the recombinant cell strains was verified by sequencing, fluorescence microscopy, flow cytometry and Western blot. Results The construction of ATP11A and ATP11B cloning vectors was confirmed by gel electrophoresis and was analyzed by endonuclease. The efficiency of transfection was observed qualitatively under microscope, detected quantitatively by flow cytometry as 38.9%,41.9% respectively, and the tagged proteins were detected by Western blot to present only in the positive groups. Conclusions The expression vectors of human ATP11A and ATP11B are successfully constructed and their expression levels could be effec-tively upregulated,which lay a foundation for future mechanism studies involved to ATP11A or ATP11B.

Keywords

• atp11a|atp11b|over-express|molecular clone