【作者】 黄美容；

【导师】 胥文春；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2011， 硕士

【摘要】 脂蛋白相关磷脂酶A2(lipoprotein–associated phospholipase A2,LP-PLA2)属于VIIA型磷脂酶A2,它可以预测与冠脉事件有关的动脉粥样硬化和中风危险。目前,在国内,LP-PLA2临床诊断试剂主要依赖于从国外进口,所需费用昂贵,限制了该检测项目在我国的推广及应用,因此,研发LP-PLA2体外免疫诊断试剂尤为重要。目的本研究旨在获取人脂蛋白相关磷脂酶A2蛋白,制备相应抗体,同时验证经生物信息学预测的表位肽的抗原性,为LP-PLA2体外免疫诊断试剂的研发奠定基础。方法采用Trizol法从分化的THP-1细胞中提取总RNA,经RT-PCR扩增目的片段LP-PLA2;利用分子克隆技术构建原核表达质粒;转化入大肠杆菌BL21(DE3),优化诱导表达条件,进行多步骤纯化并除去标签,获得目的蛋白,免疫新西兰大白兔,制备抗LP-PLA2的多克隆抗体;运用多种生物信息学方法对LP-PLA2抗原表位进行预测,并用纯化的LP-PLA2多克隆抗体鉴定其抗原性。结果1.成功构建了PET-28a(+)-LP-PLA2,PGEX-4T-2-LP-PLA2,pCold TF-LP-PLA2等重组表达质粒。2.经IPTG诱导表达后,SDS-PAGE电泳显示:LP-PLA2在PET-28a(+)-LP-PLA2表达载体中不表达;在PGEX-4T-2-LP-PLA2表达载体中少量表达,且主要以包涵体形式存在;在pCold TF-LP-PLA2表达载体中,获得表达量较高的融合蛋白,主要以上清形式存在。3.将pCold TF-LP-PLA2诱导表达上清经镍柱亲和层析纯化后,获得TF-LP-PLA2重组蛋白,将该重组蛋白用HRV-3C prolease酶切后,用蓝胶分离纯化,获得了纯度约为90%、分子量约为45kD的蛋白,与预期相符,且该蛋白能够被市售的LP-PLA2多克隆抗体识别,证明获得了纯度较高的LP-PLA2目的蛋白。4.制备了抗LP-PLA2的多克隆抗体,抗体效价>1:5.12×106 ,Western blot结果表明,该抗体特异性较高。5.运用生物信息学软件综合分析预测了LP-PLA2的三段表位肽,体外抗原抗体免疫反应实验证实了三段表位肽均具有良好的抗原性。结论成功表达并纯化获得了高纯度的LP-PLA2蛋白;制备了效价和纯度均较高的LP-PLA2多克隆抗体;运用生物信息学分析方法预测了LP-PLA2的三段表位肽,并证实了其具有良好的抗原性,可用于进一步制备多克隆和单克隆抗体。本研究为LP-PLA2体外诊断试剂的研发奠定了基础。更多还原

【Abstract】 Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a key enzyme as a risk predictor involved in atherosclerosis and ischemic stroke, and belongs to group VIIA of the phospholipase. At present, the clinical diagnostic reagents of LP-PLA2 mainly are dependent upon importation from foreign countries. Then the cost required is expensive, which is limited the application of LP-PLA2 in china. Therefore, it is important for us to development the immunoassays diagnostic reagents of LP-PLA2.Objective The purpose of this study was to obtain human lipoprotein-associated phospholipase A2 protein and its antibody which was used to verify antigenic epitopes of LP-PLA2 predicted by bioinformatics. This study will afford a foundation for further establishing the immunoassays of LP-PLA2.Methods Total RNA was isolated from differentiated THP-1 cells, and LP-PLA2 gene was amplified by RT-PCR. The PCR fragments were then cloned into expression vector, following by sequencing analysis. The positive recombinant plasmid was transformed into E.coli BL21(DE3) and the recombinant protein LP-PLA2 was expressed, Then the purified protein was identified by SDS-PAGE and Western blot. Rabbits were immunized with the LP-PLA2 protein and the polyclonal antibody was obtained. At the same time, predicted epitope of LP-PLA2 using variety of bioinformatics methods, and the antigenicity was identified with the LP-PLA polyclonal antibodies.Results 1. LP-PLA2 gene was successfully cloned into Prokaryotic expression vector PET-28a(+)-LP-PLA2,PGEX-4T-2-LP-PLA2,and pCold TF-LP-PLA2.2. After expression induced by IPTG, SDS-PAGE results showed that there was no LP-PLA2 protein expressed in the PET-28a(+)-LP-PLA and a small quantities of LP-PLA2 recombination protein were found expressed in the PGEX-4T-2-LP-PLA2 existing in the form of inclusion bodies. Besides there was LP-PLA2 recombination protein expressed in the pCold TF-LP-PLA2,existing as soluble protein of E. coli.3. After LP-PLA2 expression supernatants were purified by Ni affinity chromatography and blue gel affinity chromatography respectively, the obtained LP-PLA2 protein which could reach a purity of about 90% was found to exist as a protein of molecular weight of about 45kD, and was identified by commercial polyclonal antibodies. reach 1:5.12×106 by ELISA. Then the specificity was confirmed by Western blot.5. Three epitope peptides of LP-PLA2 were predicted by bioinformatics software and were confirmed having good antigenicity by antigen-antibody immune response in vitro experiments. Conclusion The LP-PLA2 proteins and the specific polyclonal antibodies have been obtained. Three sections epitope peptides of LP-PLA2 were predicted by bioinformatics software and then confirmed of good antigenicity by antigen-antibody immune response in vitro experiments. What’s more these peptides could be used for further preparing polyclonal antibodies and monoclonal antibodies. Thus this study could afford a foundation for further establishing the immunoassays of LP-PLA2.4. The polyclonal antibodies were obtained and titers examined could更多还原