Fusion Expression And Functional Characterization Of Recombinant Human Interleukin10

Objective: Interleukin 10 is a kind of protein secreted by mononuclear. Its comparative molecular weight is 21000Da. It has all kinds of biologic activity. It can inhibit the secretion of mononuclear cell and the synthetion of infiamation cell, which exerts important effect in the cure of disease such as tumor , virosis, and cardiovascular disease. In our reseach we counstructed syncretic protein molecular of recombinant human interleukin 10 and obtained purified rhIL-10 by one step purification. So that we could further study the mechanism of IL-10 hi some diseases.Methed:(1) The gene sequence of IL-10 was amplified by RT-PCR technique. Product of PCR was subcloned. Transform positive constructions into BL21 (DE3) pLyse cells. The fusion protein was conformed by 12% SDS-PAGE and western blot.(2) After cell broking , the inclusion body was pelleted by centrifugation, dissolves in 7M urea, then purified in one step by Ni column(Ni2+-sepharose 6B). The purified rhIL-10 was refolded by diluted pluration under low temperature.(3) Peripheral blood mononuclear cells (PBMCs) were divided into experiment group and control group, then different concentration refolded rhIL-10 were added into experiment group and control group respectively. The contents of TNF-a were determined by ELASA.So that we can investigate the inhibition of rhIL-10 on PBMCs.Results:(1) Plasmid of rhIL-10 was succeccfully constructed. It was transformed into BL21 (DE3) pLyse cells. By the induction of 1 mMIPTG, fusion protein was mainly expressedin the form of inclusion body(2) The purity of rhIL-10 was 80.42 % and the recovery rate was 66.72 %. The purified rhIL-10 was diluted with the maturation buffer and dialyzed against 0. 02M PB buffer.(3)The refolded rhIL-10 may inhibit TNF-a secreted by mononuclear cells.Conclusion:Plasmid of IL-10 was succeccfully constructed. PCR甌7/NT-TOPO?expression vector was effectively expressed in BL21 (DE3) pLyse cells. After the purified rhIL-10 was refolded, the plural rhIL-10 may inhibit TNF secreted by mononuclear cells.