关键词: CD74, 杆状病毒, 昆虫细胞, 蛋白表达

Abstract: Objective To establish an expression system of human II CD74 extracellular fragment by insect baculovirus vector expression system in insect cells. Methods hCD74 full extracellular fragment (73-232 aa) and the core area combined with MIF (109-192 aa) were amplified by PCR, and inserted into pFastBacDual vector placed promoter of PPH. Recombinant plasmid was transformed into E. coli DH10Bac and obtained recombinant virus bacmid by site specific transposition. Recombinant bacmid was transfected Sf-21 cell line. The target mRNA transcription were tested by RT-PCR and recombinant protein expression were analyzed by SDS-PAGE and Western-blot. Results The full extracellular fragment (73-232 aa) of hCD74 could be expressed in insect cells and the target protein can be detected using antibodies of His tag and CD74. In addition, it can achieve the maximum expression in cells after 72h infection and inoculation using the virus titer MOI 4. The core area of 109-192 aa fragment detected the mRNA transcription, but not achieve the protein expression. Conclusion hCD74 extracellular fragment was successfully expressed in Sf-21 cell, and optimized the best protein expression conditions.

Key words: CD74, baculovirus, insect cell, protein expression