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

Objective (1) To investigate the relationships between the +1444C/T polymorphism in C-reactive protein gene and the concentration of C-reactive protein and the risk of coronary heart diseases(CHD). (2) To develop a prokaryotic expression, purification system of recombinant E. coli which can express C-reaction protein (CRP) and detect the purified protein with Western Blotting method.Methods (1) Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to analyze the frequency distribution of CRP +1444 C/T genotypes and alleles in samples of 128 coronary heart disease (coronary stenosis more than 50%) and 119 unrelated normal individuals. The serum CRP and lipid levels of the objects were also measured. (2) 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. The purified protein was detected with Western Blotting method.Results (1) We detect +1444C/T polymorphism in our study. The frequencies of CC, CT genotypes of +1444C/T were 89.1%, 10.9%, 89.9%, 10.1%, patients and controls respectively. The frequencies of allele C were 94.5%, 95.0%, and the frequencies of allele T were 5.5%, 5.0%, patients and controls respectively. There were no significant differences in frequencies of genotype and allele between controls and CHD patients (P>0.05), but the concentrations of CRP in different genotype and allele subgroups of controls have significant difference (P<0.05). And in CHD patients, CRP, TC, TG, LDL-C, Lp(a) were higher in than those in controls (P<0.05). (2) 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 gotten. The results of Western Blotting suggest that the purified protein has single antigenicity.Conclusions (1) The CRP gene +1444C/T variant influences basal CRP level innormal people, but was not associated with CHD. These findings have implications both for the prediction and pathogenesis of coronary heart disease. There may eventually be a need to establish genotype-specific risk thresholds of CRP in the prediction of CHD risk and allow clarification of the genetic link between CRP and many diseases. (2) Recombinant human CRP was expressed at a high level 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.