LP-PLA2的原核表达、抗体制备及表位分析
发布时间：2018-11-25 11:43
【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。
[Abstract]:Lipoprotein associated phospholipase A 2 (lipoprotein associated phospholipase A 2) belongs to VIIA type phospholipase A 2, which can predict the risk of atherosclerosis and stroke associated with coronary events. At present, the LP-PLA2 clinical diagnostic reagent mainly depends on imported from abroad, and the cost is expensive, which limits the application and popularization of the test project in China. Therefore, it is particularly important to develop LP-PLA2 in vitro immunodiagnostic reagent. Objective to obtain human lipoprotein associated phospholipase A2 protein and prepare the corresponding antibody, and to verify the antigenicity of the epitope peptide predicted by bioinformatics, so as to lay a foundation for the development of LP-PLA2 immunological diagnostic reagent in vitro. Methods the total RNA, was extracted from differentiated THP-1 cells by Trizol method and the target fragment LP-PLA2; was amplified by RT-PCR. The prokaryotic expression plasmid was constructed by molecular cloning technique. E. coli BL21 (DE3) was transformed into Escherichia coli (DE3), and the expression conditions were optimized. The target protein was purified and the label was removed. The target protein was immunized against New Zealand white rabbits, and the polyclonal antibody against LP-PLA2 was prepared. LP-PLA2 epitopes were predicted by various bioinformatics methods and their antigenicity was identified with purified LP-PLA2 polyclonal antibodies. Result 1. PET-28a ()-LP-PLA2,PGEX-4T-2-LP-PLA2,pCold TF-LP-PLA2 and other recombinant expression plasmids were successfully constructed. 2. After IPTG induced expression, SDS-PAGE electrophoresis showed that LP-PLA2 was not expressed in PET-28a ()-LP-PLA2 expression vector, but was expressed in a small amount in PGEX-4T-2-LP-PLA2 expression vector, and mainly in the form of inclusion body. In the pCold TF-LP-PLA2 expression vector, the fusion protein with high expression level was obtained, and the main supernatant was found. 3. The recombinant protein of pCold TF-LP-PLA2 was purified by nickel column affinity chromatography. The recombinant protein was digested with HRV-3C prolease and purified with blue gel. The purity of the recombinant protein was about 90%. The protein with a molecular weight of about 45kD was in line with the expectation, and the protein could be recognized by the LP-PLA2 polyclonal antibody on the market. It was proved that the purified LP-PLA2 target protein was obtained. 4. The polyclonal antibody against LP-PLA2 was prepared. The titer of the antibody was 1: 5.12 脳 10 ~ 6, Western blot. The specificity of the antibody was high. 5. The three-segment epitope peptides of LP-PLA2 were predicted by bioinformatics software. The results of antigen-antibody immunoreactivity in vitro confirmed that all three epitope peptides had good antigenicity. Conclusion High purity LP-PLA2 protein was successfully expressed and purified, and LP-PLA2 polyclonal antibody with high titer and purity was prepared. The three-segment epitope peptide of LP-PLA2 was predicted by bioinformatics, and its antigenicity was confirmed. It can be used to prepare polyclonal and monoclonal antibodies. This study laid a foundation for the development of LP-PLA2 in vitro diagnostic reagent.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：2355979
[Abstract]:Lipoprotein associated phospholipase A 2 (lipoprotein associated phospholipase A 2) belongs to VIIA type phospholipase A 2, which can predict the risk of atherosclerosis and stroke associated with coronary events. At present, the LP-PLA2 clinical diagnostic reagent mainly depends on imported from abroad, and the cost is expensive, which limits the application and popularization of the test project in China. Therefore, it is particularly important to develop LP-PLA2 in vitro immunodiagnostic reagent. Objective to obtain human lipoprotein associated phospholipase A2 protein and prepare the corresponding antibody, and to verify the antigenicity of the epitope peptide predicted by bioinformatics, so as to lay a foundation for the development of LP-PLA2 immunological diagnostic reagent in vitro. Methods the total RNA, was extracted from differentiated THP-1 cells by Trizol method and the target fragment LP-PLA2; was amplified by RT-PCR. The prokaryotic expression plasmid was constructed by molecular cloning technique. E. coli BL21 (DE3) was transformed into Escherichia coli (DE3), and the expression conditions were optimized. The target protein was purified and the label was removed. The target protein was immunized against New Zealand white rabbits, and the polyclonal antibody against LP-PLA2 was prepared. LP-PLA2 epitopes were predicted by various bioinformatics methods and their antigenicity was identified with purified LP-PLA2 polyclonal antibodies. Result 1. PET-28a ()-LP-PLA2,PGEX-4T-2-LP-PLA2,pCold TF-LP-PLA2 and other recombinant expression plasmids were successfully constructed. 2. After IPTG induced expression, SDS-PAGE electrophoresis showed that LP-PLA2 was not expressed in PET-28a ()-LP-PLA2 expression vector, but was expressed in a small amount in PGEX-4T-2-LP-PLA2 expression vector, and mainly in the form of inclusion body. In the pCold TF-LP-PLA2 expression vector, the fusion protein with high expression level was obtained, and the main supernatant was found. 3. The recombinant protein of pCold TF-LP-PLA2 was purified by nickel column affinity chromatography. The recombinant protein was digested with HRV-3C prolease and purified with blue gel. The purity of the recombinant protein was about 90%. The protein with a molecular weight of about 45kD was in line with the expectation, and the protein could be recognized by the LP-PLA2 polyclonal antibody on the market. It was proved that the purified LP-PLA2 target protein was obtained. 4. The polyclonal antibody against LP-PLA2 was prepared. The titer of the antibody was 1: 5.12 脳 10 ~ 6, Western blot. The specificity of the antibody was high. 5. The three-segment epitope peptides of LP-PLA2 were predicted by bioinformatics software. The results of antigen-antibody immunoreactivity in vitro confirmed that all three epitope peptides had good antigenicity. Conclusion High purity LP-PLA2 protein was successfully expressed and purified, and LP-PLA2 polyclonal antibody with high titer and purity was prepared. The three-segment epitope peptide of LP-PLA2 was predicted by bioinformatics, and its antigenicity was confirmed. It can be used to prepare polyclonal and monoclonal antibodies. This study laid a foundation for the development of LP-PLA2 in vitro diagnostic reagent.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
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