C反应蛋白单体的制备,鉴定及其初步应用研究

Preparation,Identification and its Clinical Application of Monomeric CRP

C反应蛋白单体参与了机体的炎性反应,在多种无菌性炎症及动脉粥样硬化形成过程中起着重要作用,因此获得高纯度具有免疫原性的C反应蛋白单体(Monomeric CRP,mCRP)对于mCRP的诊断及指导靶向mCRP的治疗具有重要的临床应用价值。研究将CRP五聚体(Pentameric CRP,pCRP)在解离缓冲液(40 mmol/L Tris+500 mmol/L NaCl+2 mmol/L EDTA+8 mol/L脲)中解离60 d后采用Superdex 200 Increase10/300G分子筛分离解离产物,利用SDS-PAGE电泳及免疫印迹(WB)鉴定mCRP蛋白的纯度及免疫原性,化学发光免疫分析法研究急性心肌梗死患者血清样品中mCRP与pCRP交叉反应性;SDS-PAGE及WB结果显示pCRP在解离缓冲液中解离60 d后可部分解离为mCRP,进一步采用分子筛分离后分段收集分离产物,SDS-PAGE及WB显示解离获得的mCRP在相对分子质量约23000位置见单一明显条带,提示采用解离+分子筛方法可获得高纯度具有免疫原性的mCRP,随后作者采用化学发光免疫分析法建立的检测体系提示获得的mCRP抗原与mCRP单克隆抗体具有良好的反应性,交叉反应试验提示mCRP与pCRP不具有交叉反应性。本研究提示采用解离法结合分子筛技术可以获得具有免疫原性高纯度的mCRP蛋白,并可用于临床mCRP的定量检测。

The monomeric C-reactive protein(monomeric CRP,mCRP)plays a role in the body's inflammatory response and has an important function in various sterile inflammations and the formation of atherosclerosis.Therefore,obtaining a high-purity immunogenic monomeric CRP has significant clinical application value in diagnosis and treatment of mCRP related disease.The pentameric CRP(pCRP)was dissociated in a dissociation buffer(40 mmol/L Tris+500 mmol/L NaCl+2 mmol/L EDTA+8 mol/L urea)for 60 days,after which the dissociation products were separated using Superdex 200 Increase10/300G gel filtration.The purity and immunogenicity of the mCRP protein were identified using SDS-PAGE electrophoresis and Western blotting(WB).The chemiluminescence immunoassay was used to study the cross-reactivity of mCRP and pCRP in serum samples from patients with acute myocardial infarction.Results from SDS-PAGE and WB showed that pCRP can be partially dissociated into mCRP in the dissociation buffer after 60 days.After further gel filtration separation and segment collection of the separated products,SDS-PAGE and WB showed that the dissociated mCRP appeared as a single prominent band at around 23 ku,indicating that the combination of dissociation and gel filtration can yield a high-purity immunogenic mCRP.Subsequently,the authors detection system established using the chemiluminescence immunoassay suggested that the obtained mCRP antigen has good reactivity with the mCRP monoclonal antibody.Cross-reactivity tests indicated that mCRP and pCRP do not have cross-reactivity.This research suggests that the combination of dissociation methods and gel filtration technology can obtain a high-purity immunogenic mCRP protein,which can be used for the quantitative detection of clinical mCRP.