Expression of mannose-binding lectin associated protein 19(MAp19) in HeLa cells
LI Ting;LI Ping;JIA Tianjun;JIA Xiaohui;ZHAO Xia;CHENG Yongting;Research Institute of Pathogen Biology and Immunology,Key Laboratory of Clinical Diagnostics,Hebei North University;
Objective To construct an eukaryotic expression vector of mannose-binding lectin associated protein 19( MAp19) and further express MAp19 fusion proteins. Methods MAp19 gene fragment was amplified by PCR. The eukaryotic expression vector pc DNA3. 1 / Myc-His A-MAp19 was constructed by gene cloning technology,identified by restriction enzyme digestion and further confirmed by sequencing. The recombined plasmid was then transfected into He La cells by liposome-mediated method,and the positive clones with vector pc DNA3. 1 / Myc-His A-MAp19 were selected by G418.Location of MAp19 fusion proteins in the transfected He La cells was observed under the fluorescence microscope,and the expression levels of MAp19 fusion proteins were detected by Western blotting. Results The recombined vector pc DNA3. 1 /Myc-His A-MAp19 was successfully constructed. The result of DNA sequencing was in accordance with NCBI data bank. By G418 selecting,we obtained the He La cells which could stably express exogenous MAp19 fusion proteins. The protein was located in the cytoplasm. Western blotting also suggested that the MAp19 was secretory protein. Conclusion The vector expressing MAp19 has been prepared successful y,and can express the target protein( MAp19) in the eukaryotic cel s( He La cel s).