ABSTRACT
Objective To express VP1recombinant protein of Echovirus 30 (ECHO30) in E.coli BL21(DE3) and to develop an enzyme-linked immunoassay (EIA) based on the recombinant antigen for detecting antibodies to ECHO30.Methods The target VP1gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR).The PCR products of the gene were inserted into pET44a vector,and then expressed in E.coli BL21( DE3 ).The purified recombinant protein was used for the development of EIA.Results The molecular weight of the recombinant protein was 95 000,and the antigenicity of which was identified by Western blot and EIA.Conclusion The recombinant protein VP1has been successfully expressed and purified,which can be used as diagnostic antigen.
ABSTRACT
Objective To investigate the expression of Toll-like receptor 2 and 4 (TLR2 and TLR4) in peripheral blood mononuclear cells (PBMCs) and the serum inflammatory cytokine levels in patients with liver failure. Methods The expressions of TLR2 mRNA and TLR4 mRNA in PBMCs were detected by RT-PCR in 20 healthy controls, 20 chronic hepatitis B (CHB) patients, 18 liver failure patients in early stage and 14 in intermediate-end stage. The serum contents of endotoxin, TNF-α and IL-6 were detected by ELISA. Results Compared with the healthy controls, the expression of TLR2 mRNA and TLR4 mRNA, and the contents of endotoxin, TNF-α and IL-6 increased in CHB patients and liver failure patients ( both early stage and intermediate-end stage) ( F = 32.997, 37.476, 23. 951,57. 265 and 38. 403, P < 0.01 ). TLR2 mRNA expression in liver failure patients in intermediate-end stage was higher than that in the early stage, but that for TLR4 mRNA was lower than that in early stage. The expressions of TLR2 mRNA and TLR4 mRNA in PBMCs were significantly correlated with the contents of TNF-α and IL-6 ( r = 0. 917 and 0. 788, P < 0. 01 ). Conclusion The inflammation reaction mediated by TLR2 and TLR4 might participate in the pathogenesis of liver failure.