【作者】 赵磊；

【导师】 周新； 钱士匀； 郑芳；

【作者基本信息】 武汉大学 ， 临床检验诊断学， 2005， 硕士

【摘要】 目的 (1)探讨C-反应蛋白+1444C/T基因多态性与C-反应蛋白(C-reactive protein,CRP)水平及冠心病(coronary heart disease,CHD)的关系。(2)原核表达C-反应蛋白的表达质粒,并对目的蛋白进行纯化,验证纯化产物的生物学活性。 方法 (1)采用聚合酶链反应—限制性片段长度多态性(PCR-RFLP)技术对128例冠心病患者(至少1支冠状动脉直径狭窄≥50%)和119名湖北地区汉族正常人的CRP+1444C/T基因型和等位基因频率进行了检测,同时测定血清超敏C-反应蛋白(hsCRP)及血脂水平。分析不同基因型之间的血清CRP含量的差异。(2)在大肠杆菌中表达质粒CRP-pCRT7/NT,用氨苄青霉素平板筛选转化子,用IPTG诱导表达,表达产物经SDS-PAGE分析,并利用组氨酸亲和层析柱纯化,Western Blotting验证纯化产物抗原活性。 结果 (1)湖北地区汉族人群中存在CRP+1444C/T多态性,在冠心病组中基因型分布为:CC型89.1%,CT型10.9%,对照组分别为89.9%和10.1%。冠心病组中等位基因频率C和T分别为94.5%与5.5%,对照组中分别为95.0%与5.0%。冠心病组和对照组之间的基因型频率和等位基因频率均无显著性差异(P>0.05),但血清CRP水平在对照组不同基因型之间有显著性差异(P>0.05),正常个体中CT杂合子携带者的CRP水平比CC型纯合子携带者的高,但本研究并未发现正常组与CHD组之间CRP基因+1444C/T的基因型和等位基因的频率存在显著性差异,提示该基因变异可能不是中国人群CHD发病的易感因素。冠心病组的血清TC、TG、LDL-C、Lp(a)显著高于对照组,而两组间HDL-C、ApoAI、ApoB的差异无显著性。(2)成功地在大肠杆菌表达系统中表达出含6个组氨酸尾的CRP融合蛋白,在上SDS-PAGE表现出一条约30kD的阳性条带,经组氨酸亲和层析柱纯化后,获得CRP的蛋白纯品,Westen Blotting实验证实其确实具有CRP的抗原性。 结论 (1)本研究首次报道中国汉族人群C-反应蛋白(C-reactive protein,CRP)+1444C/T基因多态性与CRP水平、血清脂质水平及冠心病(CHD)的关系,将有助于建立用于疾病预测的基于CRP基因型特异性的风险阈值,并在遗传学上澄清CRP同多种疾病之间的联系。(2)CRP可在大肠杆菌中以包涵体更多还原

【Abstract】 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.更多还原