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Expression of hCD74 and MIF Truncated Fragment in Baculovirus Expression Vector System

Author: ShiWenJuan
Tutor: WangHeng;XuXueMei;SongGuoXing
School: Peking Union Medical College , China
Course: Pathogen Biology
Keywords: Baculovirus insect cell CD74 MIF protein expression
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 29
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Abstract


Human type II CD74 as the receptor of macrophage migration inhibitory factor (MIF) could interact with human-derived MIF(hMIF) and other MIF homologues derived from parasites, in connection with a variety of diseases of human beings and pathogenesis of parasites infection. Initially prokaryotic system was used for expression of those genes above to study their function, however, CD74 was not been achieved expression and purification successfully, and the expressed MIF protein is required by C8 reversed phase column to remove endotoxin, which limits the further study of these genes’mechanism of function in malaria. Baculovirus expression vector system (BEVS) is a eukaryotic expression system and has been used widely, and the genes refered above had not been studied in BEVS.By establishing a platform of baculovirus expression system, these genes have been expressed in succession. As a result, 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 8. But The core area of 109-192 aa fragment is detected the mRNA transcription, but not achieve the protein expression. hMIF and P.falciparum MIF(PfMIF) are also achieved expression in insect cells, the target proteins can be recognized by both His tag antibody and MIF-specific antibody, with PfMIF achieving the maiximum expression level after cells infected 72h. In addition, by using the binary vector pFastBacDual, we have achieved co-expression of CD74 extracellular truncated fragment (73-232 aa) with hMIF or PfMIF, respectively.In this study, we have established baculovirus expression vector system, and provides a new way for CD74 expressing and lays a solid foundation for further research of interaction between PfMIF and CD74, even other possible proteins. It also provides a technical support for expressing other interest proteins with expressed difficulty in E. coli.

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