Construction Of Eukaryotic Expression Vector Of PEGFP-C1-MyD88and Its Expression In HEK293Cells

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that play a crucial role in host cell prevention to microbial pathogens through recognizing pathogen associated molecular patterns(PAMP) and activating immunocyte. Myeloid differentiation factor88(MyD88) is known to play a central role in signaling transduction pathways of TLR and IL-1R familiy as an adapter protein. MyD88consists of an N-terminal death domain (DD), mediated domain(ID) and a C-terminal Toll/Interleukin-1receptor related domain(TIR). The TIR domain of MyD88can interact with the TIR domains of TLRs and IL-1Rs and mediate the downstream signal transduction. As a signal adapter protein, MyD88has a close contact with the developments of many diseases containing intestinal inflammation, atherosclerosis, myocardial hypertrophy and cancer. The structure and function research of MyD88will provide new direction and ideas for the treatment of these diseases.Full length ORF sequence of buffalo MyD88gene was amplified by RT-PCR method and encoded into pEGFP-Cl plasmid. After identified by colony PCR, endonuclease digestion and sequencing, the constructed pEGFP-Cl-WyD88eukaryotic recombinant expression vector was transfected into HEK293cells with liposome for transient expression. And the HEK293cell line expressing the EGFP-MyD88fusion protein stably was established by G418selection. Under fluorescent microsope, green fluorescence distributed heterogeneous in the entire cell transfected with the pEGFP-Cl-MyD88recombinant expression vector for48hour, whereas it distributed homogeneously in the cell transfected with the pEGFP-Cl empty vector. A62kDa fusion protein special antibody binding band was observed by Western Blot detection. The positive clone expressing fusion protein stably was obtained through continued G418selection. FCM analysis showed that the positive rates of cell line transfected with empty vector and recombinant vector were93.35%and84.33%respectively. HEK293cell line expressing MyD88protein stably was constructed successfully in the study, and it lays a foundation for diseases treatment and function research about MyD88.