Expression of mouse calreticulin missing endoplasmic reticulum retain signal peptide in B16-F1 cell line
AIM: To clone mouse calreticulin (CRT) in which the endoplasmic reticulum retain signal peptide of CRT (KDEL) is removed and to construct a B16-F1 cell line which can express secretory CRT. METHODS: Mouse CRT cDNA without KDEL-encoding sequence was obtained by PCR mutagenetic techniques and cloned into the eukaryotic expression vector.The vector was then transfected into B16-F1 cells by using liposome and the single clones were selected with G418. The cell line that is successful in transfection andsecretory expression of CRT were identified by PCR and Western Blotting assay. RESULTS: Eukaryotic expression plasmid of secretory CRT was constructed successfully. After transfected into B16-F1 cells, secretory CRT could be expressed and released into the cell medium. CONCLUSION:By removing the KDEL sequence, CRT losted its ability of retaining in endoplasmic reticulumand was released into cell medium as a secretory protein. This study explored a new way for CRT releasing into extracellular environment and offered a new research tool for anti-tumor immunotherapy.