A Study On The Relationships Between C-reactive Protein And Coronary Heart Disease

Objective: (1)To investigate the relationships between the Taq polymorphism in interieukin-1β gene and the risk of coronary heart diseases(CHD) and the concentration of C-reactive protein. (2) To develop an expression, purification system of recombinant E. coli which can express C-reaction protein (CRP).Methods: (i) The genotype and allele frequencies of interleukin-1p gene polymorphism were idenitified by polymerase chain reaction(PCR)- restriction fragment length polymorphism (RFLP) in 78 CHD patients and 130 healthy people. (2) The CRP cDNA was amplified by PCR from human liver cDNA library and inserted into expression vector pCRT7/NT. The recombined plasmid CRP-pCRT7/NT which expressed the fusion protein of CRP was then transferred into lysogenic host strain E coli. BL21(DE3). The target protein was identified using SDS-polyacrylamide gel electrophoresis(SDS-PAGE). Affinity chromatography was used for protein purification.Results: (1) The C allele frequency of interleukin-1β gene polymorphism in controls and CHD patients were 0.942, 0.965 respectively, and T allele frequency were 0.058, 0.035 respectively; There were no significant differences in frequencies of genotype and allele between controls and CHD patients(P>0.05), but the concentrations of C-reactive protein in different genotype and allele subgroups have significant differences(P<0.05). (2) A plasmid which expressed CRP protein was constructed and CRP was successfully expressed in E coli. BL21(DE3) with a 6 histamine tag; The purity of recombinant protein was detected by SDS-PAGE as a single band at 30kD approximately. Using histamine affinity chromatography purification system the purified CRP was gained.Conclusion: (1)These results suggested that the TaqDpolymorphism of interleukin-1β was associated with the concentrations of C-reactive protein in normal people but not with coronary heart diseases(CHD). (2) Recombinant human CRP was expressed in inclusion bodies of E coli. BL21(DE3) and keep its antigen activity which made a good foundation for the preparation of the CRP antibody and the CRP test kit.