CN112285347B - ELISA detection kit for pathogenic antibodies in pig serum sample - Google Patents
ELISA detection kit for pathogenic antibodies in pig serum sample Download PDFInfo
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- CN112285347B CN112285347B CN202011062115.5A CN202011062115A CN112285347B CN 112285347 B CN112285347 B CN 112285347B CN 202011062115 A CN202011062115 A CN 202011062115A CN 112285347 B CN112285347 B CN 112285347B
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Abstract
Description
技术领域：Technical field:
本发明属于生物技术领域，具体涉及一种猪血清样中病原抗体ELISA检测试剂盒，特别涉及分离和鉴定PRRSV抗原肽及其应用，特别是用于猪血清样中病原抗体ELISA检测的方法。The invention belongs to the field of biotechnology, and specifically relates to an ELISA detection kit for pathogenic antibodies in pig serum samples, and particularly relates to separation and identification of PRRSV antigen peptides and applications thereof, in particular to a method for ELISA detection of pathogenic antibodies in pig serum samples.
背景技术：Background technology:
宿主机体的免疫系统在对外源性的病原感染，抗原刺激或疫苗（以下统称为外源性免疫原）免疫的过程做出反应的过程中，主要通过宿主免疫系统中的抗原提呈细胞（APCs）对外源性的抗原进行捕获，并进行加工提呈，将完整的外源性免疫原蛋白降解切割为长度为12-24的氨基酸长度的抗原肽片段，以上的抗原肽片段可以装配与主要组织相容性复合物（MHC）II类分子的抗原肽结合槽中，形成“MHC-II-抗原肽”复合物。在猪体内，负责完成此过程的MHC-II类分子为猪白细胞抗原-II DR（Swine leukocyte antigen DR，SLA-DR）并表达于多种APCs表面，如猪肺泡巨噬细胞，或淋巴组织内的巨噬细胞和树突状细胞。猪APCs表面的SLA-DR分子所加工提呈的抗原肽，能够作为猪特异性CD4+T细胞表位，被猪体内相应的CD4+T细胞所识别，最终激活猪机体的适应性免疫应答，产生免疫记忆，保护机体免受病原的感染。When the host's immune system responds to exogenous pathogen infection, antigen stimulation or vaccine (hereinafter collectively referred to as exogenous immunogens), the host's immune system mainly captures exogenous antigens through antigen presenting cells (APCs) in the host's immune system, processes and presents them, and degrades and cuts the complete exogenous immunogen protein into antigen peptide fragments with a length of 12-24 amino acids. The above antigen peptide fragments can be assembled into the antigen peptide binding groove of the major histocompatibility complex (MHC) class II molecule to form an "MHC-II-antigen peptide" complex. In pigs, the MHC-II class molecule responsible for completing this process is Swine leukocyte antigen DR (SLA-DR) and is expressed on the surface of a variety of APCs, such as pig alveolar macrophages, or macrophages and dendritic cells in lymphoid tissues. The antigenic peptides processed and presented by the SLA-DR molecules on the surface of pig APCs can serve as pig-specific CD4 + T cell epitopes and be recognized by the corresponding CD4 + T cells in the pig body, ultimately activating the pig's adaptive immune response, generating immune memory, and protecting the body from pathogen infection.
因此在猪免疫系统活化的过程中，最终引发免疫应答的有效抗原成分仅仅是外源性免疫原被APCs加工提呈后长度为12-24个氨基酸的抗原肽片段（即猪特异性CD4+ T表位），而并非完整的免疫原。因此，直接使用来源于免疫原的CD4+T表位片段刺激机体即可激活机体的免疫系统，产生与使用完整外源性蛋白抗原刺激机体免疫系统类似的免疫应答。此外，使用来源于完整外源性免疫原的一个或多个CD4+T表位片段通过全人工合成或是基因工程重组表达的方法可以表达为一种仅含有CD4+T表位的重组蛋白，所获得的重组蛋白可以作为一种基因工程疫苗用于猪传染病的预防。Therefore, in the process of activating the pig immune system, the effective antigen component that ultimately triggers the immune response is only the antigen peptide fragment of 12-24 amino acids in length (i.e., pig-specific CD4+ T epitope) after the exogenous immunogen is processed and presented by APCs, rather than the complete immunogen. Therefore, directly stimulating the body with CD4 + T epitope fragments derived from the immunogen can activate the body's immune system and produce an immune response similar to that of stimulating the body's immune system with complete exogenous protein antigens. In addition, one or more CD4 + T epitope fragments derived from a complete exogenous immunogen can be expressed as a recombinant protein containing only CD4 + T epitopes through full artificial synthesis or genetic engineering recombinant expression. The obtained recombinant protein can be used as a genetically engineered vaccine for the prevention of swine infectious diseases.
猪繁殖与呼吸综合征（Porcine reproductive and respiratory syndrome，PRRS）是由猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndromevirus，PRRSV）引起的以母猪流产和仔猪呼吸障碍为主要特征的病毒性传染病，并能引起严重的免疫抑制。该病毒已在全球猪群中广泛传播，给世界养猪业造成了巨大的经济损失，已成为全球规模化猪场的主要疫病之一，也是全球猪病控制上的一大难题。市场上现有的PRRSV抗体ELISA检测试剂盒，主要以PRRSV病毒的主要核衣壳蛋白N为包板抗原进行检测，存在因为病毒抗原变异所导致的漏检现象。Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), characterized by sow abortion and piglet respiratory disorders, and can cause severe immunosuppression. The virus has spread widely in pig herds around the world, causing huge economic losses to the world's pig industry. It has become one of the main epidemic diseases in large-scale pig farms around the world and a major problem in global pig disease control. The existing PRRSV antibody ELISA detection kits on the market mainly use the main nucleocapsid protein N of the PRRSV virus as the coating antigen for detection, and there is a phenomenon of missed detection due to viral antigen variation.
本申请以猪及猪繁殖与呼吸综合征病毒（PRRSV）为例，以体外培养的猪骨髓诱导树突状细胞（Bone Marrow derived dendritic cells, BM-DCs）作为猪特异性抗原提呈细胞（APCs），使用PRRSV感染BM-DCs模拟PRRSV感染宿主或免疫宿主后其编码的病毒抗原在猪体内加工提呈的过程，通过SLA-DR富集BM-DCs上的“SLA-DR-抗原肽”复合物，洗脱抗原肽后通过质谱技术测定来源于PRRSV不同病毒蛋白抗原肽氨基酸序列的方法，再辅以人工感染PRRSV病毒猪来源的血清样本作为阳性对照，检测所有质谱分析结果获得的抗原肽序列中可被PRRSV感染猪血清识别的有效抗原肽序列，挑选其中来源于不同PRRSV蛋白的有效抗原肽组合制备为人工抗原，替代传统PRRSV抗体ELISA检测试剂盒中的N抗原，以提高检测结果的准确性。This application takes pigs and porcine reproductive and respiratory syndrome virus (PRRSV) as an example, uses in vitro cultured porcine bone marrow derived dendritic cells (BM-DCs) as porcine specific antigen presenting cells (APCs), uses PRRSV to infect BM-DCs to simulate the process of processing and presentation of viral antigens encoded by PRRSV in pigs after PRRSV infects the host or immunizes the host, enriches the "SLA-DR-antigen peptide" complex on BM-DCs by SLA-DR, and uses mass spectrometry technology to determine the amino acid sequence of antigen peptides derived from different viral proteins of PRRSV after eluting the antigen peptides, and then uses serum samples from pigs artificially infected with PRRSV virus as positive controls to detect effective antigen peptide sequences that can be recognized by PRRSV-infected pig serum in all antigen peptide sequences obtained by mass spectrometry analysis, and selects effective antigen peptide combinations derived from different PRRSV proteins to prepare artificial antigens to replace the N antigen in the traditional PRRSV antibody ELISA detection kit to improve the accuracy of the detection results.
发明内容：Summary of the invention:
本发明的第一个目的是通过组合质谱分析技术和抗体ELISA筛选技术，提供一在猪种属上对病原或外源性蛋白上可被该病原或蛋白特异性抗体所识别的有效的特异性抗原肽(即CD4+T表位)进行筛选鉴定方法，并且以猪繁殖呼吸道综合征为例来进行说明。The first objective of the present invention is to provide a method for screening and identifying effective specific antigen peptides (i.e., CD4+ T epitopes) on pathogens or exogenous proteins that can be recognized by pathogen or protein-specific antibodies in pig species by combining mass spectrometry analysis technology and antibody ELISA screening technology, and to illustrate this method using porcine reproductive and respiratory syndrome as an example.
本发明的第二个目的以第一个目的中所获得的可被相应病原或蛋白特异性抗体所识别的抗原肽，通过组合串联后再进行基因表达，替代原始病原或抗原，用于ELISA检测，以提高检测的准确度。The second purpose of the present invention is to use the antigenic peptides obtained in the first purpose that can be recognized by the corresponding pathogen or protein-specific antibodies, combine them in series and then express them genetically, so as to replace the original pathogen or antigen for ELISA detection to improve the detection accuracy.
一种猪血清样中病原抗体ELISA检测试剂盒，其特征在于，所述试剂盒包括猪病原特异性抗原肽，所述抗原肽是利用猪源APCs在猪种属上对病原或外源性蛋白进行特异性抗原肽(即CD4+T表位)筛选鉴定的方法得到，所述方法包括如下步骤：An ELISA kit for detecting pathogen antibodies in pig serum samples, characterized in that the kit comprises pig pathogen-specific antigen peptides, the antigen peptides are obtained by screening and identifying specific antigen peptides (i.e., CD4+T epitopes) of pathogens or exogenous proteins on pig species using pig-derived APCs, and the method comprises the following steps:
步骤一，猪源APCs细胞的制备；Step 1, preparation of porcine APCs cells;
步骤二，抗原肽的质谱鉴定；Step 2: mass spectrometry identification of antigenic peptides;
步骤三，基于NanoLuc融合蛋白的抗原肽的ELISA验证。Step 3: ELISA verification of antigenic peptide based on NanoLuc fusion protein.
优选的，本发明中所述猪源APCs细胞的制备采用如下方法：（1）猪骨髓源树突状细胞，通过分离仔猪的长腿骨，开孔后使用细胞培养液冲洗骨髓腔，获得的骨髓细胞经过猪粒细胞巨噬细胞-集落刺激因子（GM-CSF）的诱导后经体外培养7天后获得（如图1所示）；（2）猪肺泡巨噬细胞，通过冲洗猪肺脏后，将肺泡灌洗液离心后获得（如图2所示）；（3）猪外周血单核细胞源巨噬细胞，收集猪抗凝血，通过使用淋巴细胞分离液，从抗凝血中获得猪外周血单核细胞，加入猪粒细胞巨噬细胞-集落刺激因子（GM-CSF）和猪白介素4（IL-4）体外诱导培养7天后获得（如图3所示）；或者（4）使用外来免疫原（包括并不限于病原，疫苗，蛋白抗原等）免疫猪个体，其后，分离猪的脾脏，淋巴结，经过体外在单细胞滤网上研磨获得包含有猪内源性巨噬细胞的免疫细胞群（如图4所示）。Preferably, the porcine APCs cells described in the present invention are prepared by the following method: (1) porcine bone marrow-derived dendritic cells are obtained by isolating the long leg bones of piglets, opening a hole, and flushing the bone marrow cavity with cell culture medium. The obtained bone marrow cells are induced by porcine granulocyte macrophage-colony stimulating factor (GM-CSF) and cultured in vitro for 7 days (as shown in FIG. 1 ); (2) porcine alveolar macrophages are obtained by flushing the pig lungs and centrifuging the alveolar lavage fluid (as shown in FIG. 2 ); (3) porcine peripheral blood monocyte-derived macrophages are collected Porcine anticoagulated blood is prepared by using lymphocyte separation fluid to obtain porcine peripheral blood mononuclear cells from anticoagulated blood, and then adding porcine granulocyte macrophage-colony stimulating factor (GM-CSF) and porcine interleukin 4 (IL-4) to induce and culture the cells in vitro for 7 days (as shown in Figure 3); or (4) using foreign immunogens (including but not limited to pathogens, vaccines, protein antigens, etc.) to immunize individual pigs, and then separating the spleen and lymph nodes of the pigs, and grinding them on a single cell filter in vitro to obtain an immune cell population containing endogenous porcine macrophages (as shown in Figure 4).
优选的，本发明所述的抗原肽的质谱鉴定的方法包括如下步骤：（1）制备富集“SLA-DR-抗原肽”复合物；（2）获得抗原肽氨基酸序列；（3）使用质谱仪测定抗原肽的分子量，并通过质谱分析软件搜索病原来源蛋白氨基酸数据库的方式以确定抗原肽氨基酸序列。Preferably, the method for mass spectrometry identification of antigenic peptides of the present invention comprises the following steps: (1) preparing an enriched "SLA-DR-antigenic peptide" complex; (2) obtaining the amino acid sequence of the antigenic peptide; (3) determining the molecular weight of the antigenic peptide using a mass spectrometer, and determining the amino acid sequence of the antigenic peptide by searching the pathogenic protein amino acid database using mass spectrometry analysis software.
所述制备富集“SLA-DR-抗原肽”复合物的方法为：使用APCs经过免疫原刺激（如病毒感染，加入抗原和APCs共培养等方式），再使用细胞裂解缓冲液裂解APCs制备细胞裂解液，使用识别SLA-DR的抗体从全细胞裂解液中富集“SLA-DR-抗原肽”复合物。The method for preparing and enriching the "SLA-DR-antigen peptide" complex is: using APCs to be stimulated by an immunogen (such as virus infection, adding antigens and APCs co-culture, etc.), then using a cell lysis buffer to lyse the APCs to prepare a cell lysate, and using an antibody that recognizes SLA-DR to enrich the "SLA-DR-antigen peptide" complex from the whole cell lysate.
所述获得抗原肽氨基酸片段的方法为：将富集“SLA-DR-抗原肽”复合物使用三氟乙酸缓冲液处理，从SLA-DR上分离洗脱抗原肽，再使用截留分子量为5kD的超滤管，去除残留抗体及SLA-DR分子单体，使滤过液中仅含有抗原肽，再通过脱盐处理后，即得到所述抗原肽氨基酸片段。The method for obtaining the amino acid fragment of the antigen peptide is as follows: the enriched "SLA-DR-antigen peptide" complex is treated with a trifluoroacetic acid buffer, the antigen peptide is separated and eluted from the SLA-DR, and then an ultrafiltration tube with a molecular weight cutoff of 5kD is used to remove residual antibodies and SLA-DR molecular monomers so that the filtrate contains only the antigen peptide, and then the antigen peptide amino acid fragment is obtained after desalting.
优选的，本发明所述的基于NanoLuc融合蛋白的抗原肽的ELISA验证的方法包括如下步骤：（1）将所获的抗原肽依照其cDNA序列人工合成克隆进入pET28-NanoLucC1载体，表达为NanoLuc融合抗原肽，并使用NanoLuc抗体检测融合；（2）分别将NanoLuc融合抗原肽作为包板抗原，通过ELISA检测相应病原感染猪血清样本，ELISA检测为阳性的肽段即为有效的抗原肽，否则不是有效的抗原肽。Preferably, the ELISA verification method of the antigenic peptide based on the NanoLuc fusion protein of the present invention comprises the following steps: (1) the obtained antigenic peptide is artificially synthesized and cloned into the pET28-NanoLucC1 vector according to its cDNA sequence, expressed as a NanoLuc fusion antigenic peptide, and the fusion is detected using the NanoLuc antibody; (2) the NanoLuc fusion antigenic peptide is used as a coating antigen, and the corresponding pathogen-infected pig serum samples are detected by ELISA. The peptide segment that is positive in the ELISA test is a valid antigenic peptide, otherwise it is not a valid antigenic peptide.
本发明进一步将可被人工感染猪血清识别的多个阳性抗原肽串联后，在大肠杆菌中重组表达，所收获的人工重组蛋白即可作为所述猪病原特异性抗原肽用于相应病原感染猪血清的ELISA检测。The present invention further connects multiple positive antigen peptides that can be recognized by artificially infected pig serum in series, and then recombinantly expresses them in Escherichia coli. The harvested artificial recombinant protein can be used as the pig pathogen-specific antigen peptide for ELISA detection of corresponding pathogen-infected pig serum.
优选的所述试剂盒为猪血清样中PRRSV病原抗体ELISA检测试剂盒，所述猪病原特异性抗原肽为PRRSV抗原肽。Preferably, the kit is an ELISA kit for detecting PRRSV pathogen antibodies in pig serum samples, and the pig pathogen-specific antigen peptide is a PRRSV antigen peptide.
优选的，所述PRRSV抗原肽采用如下方法分离和鉴定得到，所述方法包括如下步骤：Preferably, the PRRSV antigen peptide is separated and identified by the following method, which comprises the following steps:
步骤一，猪源APCs细胞的制备；Step 1, preparation of porcine APCs cells;
步骤二，抗原肽的质谱鉴定；Step 2: mass spectrometry identification of antigenic peptides;
步骤三，基于NanoLuc融合蛋白的抗原肽的ELISA验证。Step 3: ELISA verification of antigenic peptide based on NanoLuc fusion protein.
优选的，所述猪源APCs细胞的制备的方法包括如下步骤：（1）使用4-6周龄仔猪腿骨，从中段使用无菌锯条锯断后，在断骨的另一端钻孔，使用含有EDTA的无血清RPMI 1640培养基冲洗骨髓腔，以获得猪骨髓细胞悬液；（2）将所获得的猪骨髓细胞悬液以300g离心力离心10分钟后弃去上清，加入红细胞裂解液在37℃培养20分钟以充分裂解红细胞，再加入2倍体积的PBS以终止反应，再次以300g离心10分钟以获得纯化的骨髓细胞；（3）将骨髓细胞进行计数，以1×107个骨髓细胞置于10mL含有10%胎牛血清的RPMI 1640培养基中，按照40ng每毫升的浓度加入猪GM-CSF进行培养，每间隔2天更换一次培养液，连续培养7天后，收集悬浮细胞，此即为作为猪源APCs的骨髓树突状细胞。Preferably, the method for preparing the porcine APCs cells comprises the following steps: (1) using a 4-6 week old piglet leg bone, sawing it from the middle section with a sterile saw blade, drilling a hole at the other end of the broken bone, and flushing the bone marrow cavity with a serum-free RPMI 1640 culture medium containing EDTA to obtain a porcine bone marrow cell suspension; (2) centrifuging the obtained porcine bone marrow cell suspension at 300 g for 10 minutes, discarding the supernatant, adding red blood cell lysis solution and culturing at 37° C. for 20 minutes to fully lyse the red blood cells, then adding 2 times the volume of PBS to terminate the reaction, and centrifuging again at 300 g for 10 minutes to obtain purified bone marrow cells; (3) counting the bone marrow cells, placing 1×10 7 bone marrow cells in 10 mL of RPMI 1640 culture medium containing 10% fetal bovine serum, adding porcine GM-CSF at a concentration of 40 ng per milliliter for culturing, replacing the culture medium every 2 days, and collecting the suspended cells after 7 consecutive days of culturing, which are the bone marrow dendritic cells used as porcine APCs.
优选的，所述抗原肽的质谱鉴定的方法包括如下步骤：（1）使用PRRSV-JXA1毒株以1MOI剂量感染1×108个APCs细胞，在病毒感染24小时后离心收集细胞，使用膜蛋白提取试剂盒提取APCs细胞膜蛋白以避免胞浆蛋白的污染，通过特异性识别SLA-DR的抗体（Purified Mouse Anti-Pig SLA-DR，BD Pharmingen™，Material Number: 553642）从APCs细胞膜蛋白中富集“SLA-DR抗原肽”复合物；（2）使用10%甘氨酸溶液从抗体上洗脱SLA-DR抗原肽复合物，再使用10%三氟乙酸（TFA）从SLA-DR抗原肽复合物上洗脱抗原肽；（3）将10%三氟乙酸（TFA）洗脱液通过截留分子量为5kD的超滤管进行过滤后去除SLA-DR分子，收集滤过液，此即为SLA-DR上洗脱的抗原肽；（4）将抗原肽滤过液脱盐冻干后，使用分子纯水重悬，在Orbitrap Fusion Lumos Tribrid质谱仪上机检测，获得抗原肽的质谱图谱，将PRRSV-JXA1所有的编码蛋白氨基酸序列设置为PRRSV蛋白序列库，使用ProteomeDiscoverer或PEAKS®Studio X软件对所获得的质谱图谱进行搜库，即可获得与PRRSV-JXA1编码蛋白匹配的抗原肽序列。Preferably, the mass spectrometry identification method of the antigen peptide comprises the following steps: (1) using the PRRSV-JXA1 strain to infect 1×10 8 APCs cells at a dose of 1 MOI, collecting the cells by centrifugation 24 hours after virus infection, extracting APCs cell membrane proteins using a membrane protein extraction kit to avoid contamination by cytoplasmic proteins, and using an antibody that specifically recognizes SLA-DR (Purified Mouse Anti-Pig SLA-DR, BD Pharmingen™, Material Number: 553642) enrich the "SLA-DR antigen peptide" complex from the APCs cell membrane protein; (2) use 10% glycine solution to elute the SLA-DR antigen peptide complex from the antibody, and then use 10% trifluoroacetic acid (TFA) to elute the antigen peptide from the SLA-DR antigen peptide complex; (3) filter the 10% trifluoroacetic acid (TFA) eluate through an ultrafiltration tube with a molecular weight cutoff of 5kD to remove the SLA-DR molecules, and collect the filtrate, which is the antigen peptide eluted from SLA-DR; (4) desalt and freeze-dry the antigen peptide filtrate, resuspend it in molecular pure water, and detect it on an Orbitrap Fusion Lumos Tribrid mass spectrometer to obtain the mass spectrum of the antigen peptide, set all the protein encoding amino acid sequences of PRRSV-JXA1 as the PRRSV protein sequence library, and use ProteomeDiscoverer or PEAKS ® Studio X software searches the database of the obtained mass spectrum to obtain the antigenic peptide sequence matching the protein encoded by PRRSV-JXA1.
优选的，所述基于NanoLuc融合蛋白的抗原肽的ELISA验证的方法包括如下步骤：（1）将所获的PRRSV-JXA1免疫肽依照其cDNA序列人工合成克隆进入pET28-NanoLucC1载体，表达为NanoLuc融合抗原肽，并使用NanoLuc抗体检测融合；（2）分别将NanoLuc融合抗原肽作为包板抗原，通过ELISA检测PRRSV-JXA1感染猪血清样本。Preferably, the ELISA verification method of the antigenic peptide based on the NanoLuc fusion protein comprises the following steps: (1) artificially synthesizing and cloning the obtained PRRSV-JXA1 immune peptide into the pET28-NanoLucC1 vector according to its cDNA sequence, expressing it as a NanoLuc fusion antigenic peptide, and using the NanoLuc antibody to detect the fusion; (2) using the NanoLuc fusion antigenic peptide as the coating antigen, respectively, and detecting the serum samples of PRRSV-JXA1 infected pigs by ELISA.
本发明还请求保护包括PRRSV抗原肽的重组蛋白，分别是PRRSV-融合肽段A，其氨基酸序列如SEQ ID NO：1所示，或者PRRSV-融合肽段B，其氨基酸序列如SEQ ID NO：2所示。The present invention also claims protection for a recombinant protein comprising a PRRSV antigen peptide, which is respectively a PRRSV-fusion peptide segment A, whose amino acid sequence is shown in SEQ ID NO: 1, or a PRRSV-fusion peptide segment B, whose amino acid sequence is shown in SEQ ID NO: 2.
基于以上技术方案，本发明具有如下优点和有益效果：Based on the above technical solution, the present invention has the following advantages and beneficial effects:
本发明通过对免疫原（病原或蛋白抗原）上的有效抗原肽序列进行筛选，去除免疫原中对机体不具有免疫活性（即不能激活机体适应性免疫应答）的无用肽段，筛选出免疫活性较强，可高效刺激机体产生免疫应答的抗原肽。抗原肽相对于原始免疫原，因其序列较短（12-24氨基酸残基），亲水性较强（需具备与SLA-DR结合的能力，疏水性肽段无法结合SLA-DR从而被提呈激活CD4+T细胞），因此易于在体外进行表达，并且可随意将不同抗原肽串联制备复合抗原肽，避免常规ELISA实验中使用单一抗原作为包板抗原所导致的漏检问题，以提高ELISA检测的敏感性和准确性。The present invention screens the effective antigen peptide sequences on the immunogen (pathogen or protein antigen), removes the useless peptide segments in the immunogen that have no immunological activity to the body (i.e., cannot activate the body's adaptive immune response), and screens out antigen peptides with strong immunological activity that can efficiently stimulate the body to produce an immune response. Compared with the original immunogen, the antigen peptide is easy to express in vitro because of its shorter sequence (12-24 amino acid residues) and stronger hydrophilicity (it must have the ability to bind to SLA-DR, and the hydrophobic peptide segment cannot bind to SLA-DR and thus be presented to activate CD4+T cells), and different antigen peptides can be arbitrarily connected in series to prepare composite antigen peptides, avoiding the problem of missed detection caused by using a single antigen as a plate antigen in conventional ELISA experiments, so as to improve the sensitivity and accuracy of ELISA detection.
附图说明：Description of the drawings:
图1：使用GM-CSF刺激诱导分化培养7天后骨髓树突状细胞形态：A：未诱导的骨髓细胞；B：GM-CSF骨髓树突状细胞BM-DCs。Figure 1: Morphology of bone marrow dendritic cells after 7 days of differentiation induced by GM-CSF stimulation: A: uninduced bone marrow cells; B: GM-CSF bone marrow dendritic cells BM-DCs.
图2：新鲜分离的猪肺泡巨噬细胞形态照片。Figure 2: Morphological photographs of freshly isolated porcine alveolar macrophages.
图3：猪外周血单个核细胞显微镜下形态。Figure 3: Microscopic morphology of porcine peripheral blood mononuclear cells.
图4：猪淋巴结及脾脏研磨去除红细胞后淋巴细胞。Figure 4: Lymphocytes from porcine lymph nodes and spleen after grinding and removal of red blood cells.
图5：抗原肽序列质谱分析代表性图谱（NSP1β来源）。Figure 5: Representative mass spectrometry analysis of antigenic peptide sequences (derived from NSP1β).
图6：荧光素酶NanoLuc融合免疫抗原肽的大肠杆菌表达示例。Figure 6: Example of E. coli expression of luciferase NanoLuc fusion immunogenic peptide.
图7：抗原肽串联后分段表达结果。Figure 7: The results of segmented expression after antigen peptide concatenation.
图8：本发明所得的ELISA检测方法与爱德士商品化监测试剂盒检测不一致3份血清样本（样本编号PC39，PC65，PC67）通过免疫荧光检测PRRSV感染的MARC-145细胞证实血清样本为PRRSV抗体阳性。Figure 8: The ELISA detection method obtained in the present invention is inconsistent with the detection of the commercial monitoring kit of IDEXX. Three serum samples (sample numbers PC39, PC65, PC67) were tested by immunofluorescence on PRRSV-infected MARC-145 cells to confirm that the serum samples were PRRSV antibody positive.
具体实施方式：Specific implementation method:
下面列出具体实施方案对本发明做进一步阐述，以使本领域技术人员可以更清楚得得知本发明的技术方案，并非对本发明的限制。The following specific implementation schemes are listed to further illustrate the present invention so that those skilled in the art can more clearly understand the technical solutions of the present invention, but they are not intended to limit the present invention.
实施例1：猪源APCs细胞的制备（以猪骨髓树突状细胞为例）Example 1: Preparation of porcine APCs cells (taking porcine bone marrow dendritic cells as an example)
1.使用4-6周龄仔猪腿骨，从中段使用无菌锯条锯断后，在断骨的另一端钻孔，使用含有EDTA的无血清RPMI 1640培养基冲洗骨髓腔，以获得猪骨髓细胞悬液。1. Use the leg bones of 4-6 week old piglets, saw them off from the middle with a sterile saw blade, drill a hole at the other end of the broken bone, and flush the bone marrow cavity with serum-free RPMI 1640 medium containing EDTA to obtain pig bone marrow cell suspension.
将所获得的猪骨髓细胞悬液以300g离心力离心10分钟后弃去上清，加入红细胞裂解液在37℃培养20分钟以充分裂解红细胞，再加入2倍体积的PBS以终止反应，再次以300g离心10分钟以获得纯化的骨髓细胞。The obtained pig bone marrow cell suspension was centrifuged at 300g for 10 minutes, and the supernatant was discarded. Red blood cell lysis solution was added and cultured at 37°C for 20 minutes to fully lyse the red blood cells. Then 2 volumes of PBS were added to terminate the reaction, and the suspension was centrifuged at 300g for 10 minutes again to obtain purified bone marrow cells.
将骨髓细胞进行计数，以1×107个骨髓细胞置于10mL含有10%胎牛血清的RPMI1640培养基中，按照40ng每毫升的浓度加入猪GM-CSF进行培养，每间隔2天更换一次培养液，连续培养7天后，收集悬浮细胞，此即为作为猪源APCs的骨髓树突状细胞，其形态如图1所示，可进行下一步实验。The bone marrow cells were counted and 1×10 7 bone marrow cells were placed in 10 mL of RPMI1640 culture medium containing 10% fetal bovine serum. Porcine GM-CSF was added at a concentration of 40 ng per milliliter for culture. The culture medium was replaced every 2 days. After 7 days of continuous culture, the suspended cells were collected. These were the bone marrow dendritic cells serving as porcine APCs. Their morphology is shown in Figure 1 and the next experiment can be carried out.
实施例2、抗原肽的质谱鉴定Example 2: Mass spectrometry identification of antigenic peptides
1.使用PRRSV-JXA1毒株以1MOI剂量感染1×108个APCs细胞，在病毒感染24小时后离心收集细胞，使用膜蛋白提取试剂盒提取APCs细胞膜蛋白以避免胞浆蛋白的污染，通过特异性识别SLA-DR的抗体（Purified Mouse Anti-Pig SLA-DR，BD Pharmingen™，Material Number: 553642）从APCs细胞膜蛋白中富集“SLA-DR抗原肽”复合物。1. PRRSV-JXA1 strain was used to infect 1×10 8 APCs cells at a dose of 1 MOI. Cells were collected by centrifugation 24 hours after virus infection. APCs cell membrane proteins were extracted using a membrane protein extraction kit to avoid contamination by cytoplasmic proteins. The "SLA-DR antigen peptide" complex was enriched from APCs cell membrane proteins using an antibody that specifically recognizes SLA-DR (Purified Mouse Anti-Pig SLA-DR, BD Pharmingen™, Material Number: 553642).
使用10%甘氨酸溶液从抗体上洗脱SLA-DR抗原肽复合物，再使用10%三氟乙酸（TFA）从SLA-DR抗原肽复合物上洗脱抗原肽。The SLA-DR antigen peptide complex was eluted from the antibody using a 10% glycine solution, and then the antigen peptide was eluted from the SLA-DR antigen peptide complex using 10% trifluoroacetic acid (TFA).
将10%三氟乙酸（TFA）洗脱液通过截留分子量为5kD的超滤管进行过滤后去除SLA-DR分子，收集滤过液，此即为SLA-DR上洗脱的抗原肽。The 10% trifluoroacetic acid (TFA) eluate was filtered through an ultrafiltration tube with a molecular weight cutoff of 5 kD to remove the SLA-DR molecules, and the filtrate was collected, which was the antigen peptide eluted from SLA-DR.
4.将抗原肽滤过液脱盐冻干后，使用分子纯水重悬，在Orbitrap Fusion LumosTribrid质谱仪上机检测，获得抗原肽的质谱图谱（代表性图谱如图5所示）。将PRRSV-JXA1所有的编码蛋白氨基酸序列设置为PRRSV蛋白序列库，使用Proteome Discoverer或PEAKS® Studio X软件对所获得的质谱图谱进行搜库，即可获得与PRRSV-JXA1编码蛋白匹配的抗原肽序列，其结果如表1所示。4. After the antigen peptide filtrate is desalted and freeze-dried, it is resuspended in molecular pure water and detected on the Orbitrap Fusion Lumos Tribrid mass spectrometer to obtain the mass spectrum of the antigen peptide (the representative spectrum is shown in Figure 5). All the amino acid sequences of the encoded proteins of PRRSV-JXA1 are set as the PRRSV protein sequence library, and the obtained mass spectrum is searched using Proteome Discoverer or PEAKS® Studio X software to obtain the antigen peptide sequence matching the PRRSV-JXA1 encoded protein, and the results are shown in Table 1.
实施例3：基于NanoLuc融合蛋白的抗原肽的ELISA验证Example 3: ELISA validation of antigenic peptides based on NanoLuc fusion protein
1.将所获的PRRSV-JXA1免疫肽依照其cDNA序列人工合成克隆进入pET28-NanoLucC1载体，表达为NanoLuc融合抗原肽，并使用NanoLuc抗体检测融合，如图6所示。1. The obtained PRRSV-JXA1 immune peptide was artificially synthesized and cloned into the pET28-NanoLucC1 vector according to its cDNA sequence, expressed as NanoLuc fusion antigen peptide, and the fusion was detected using NanoLuc antibody, as shown in FIG6 .
分别将NanoLuc融合抗原肽作为包板抗原，通过ELISA检测PRRSV-JXA1感染猪血清样本。其结果如表2所示。NanoLuc fusion antigen peptides were used as coating antigens to detect serum samples of pigs infected with PRRSV-JXA1 by ELISA. The results are shown in Table 2.
表1通过分析质谱技术获得的PRRSV-JXA1病毒全基因组免疫肽序列Table 1 PRRSV-JXA1 virus genome-wide immune peptide sequences obtained by mass spectrometry
表2 使用5份PRRSV抗体阳性血清与PRRSV抗体阴性血清对荧光素酶NanoLuc融合免疫抗原肽进行检测寻找阳性肽段Table 2 Using 5 PRRSV antibody positive sera and PRRSV antibody negative sera to detect luciferase NanoLuc fusion immune antigen peptides to find positive peptides
实施例4：使用复合PRRSV抗原肽重组表达蛋白作为检测抗原用于PRRSV感染个体血清样本检测。Example 4: Using the composite PRRSV antigen peptide recombinantly expressed protein as a detection antigen for detecting serum samples of PRRSV-infected individuals.
1.选取挑选的抗原肽序列，通过G4S柔性连接肽间隔的方式，设计为重组PRRSV抗原，命名为融合肽段A和B，其序列分别为SEQ ID NO：1和SEQ ID NO：2，并进行重组表达，其序列及表达结果如图7所示1. The selected antigen peptide sequences were selected and designed into recombinant PRRSV antigens by means of G4S flexible connecting peptide intervals, named fusion peptide segments A and B, whose sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and recombinant expression was performed. The sequences and expression results are shown in FIG7
2.收集100分场地猪血清样本，使用重组PRRSV抗原作为包板抗原进行ELISA检测，其检测结果如表3所示，并使用爱德士公司PRRSV X3 Herd Check试剂盒进行检测作为对照检测符合率，其结果如表4所示2. Collected 100 pig serum samples from the site, and used recombinant PRRSV antigen as the plate antigen for ELISA testing. The test results are shown in Table 3. The PRRSV X3 Herd Check kit from IDEXX was used as a control test for compliance rate. The results are shown in Table 4.
3.与爱德士公司ELISA试剂盒对照结果显示，存在三份血清样本结果不一致，其中以本发明建立的方法检测显示为阳性，爱德士公司ELISA试剂盒为阴性其结果如表4所示。进一步通过免疫荧光，使用稀释血清样本检测PRRSV感染的Marc-145细胞，确定三份样本为PRRSV抗体阳性样本，与本发明建立的方法相一致。免疫荧光结果如图8所示。3. The results of the comparison with the ELISA kit of IDEXX showed that there were three serum samples with inconsistent results, of which the results were positive when tested by the method established by the present invention, and negative when tested by the ELISA kit of IDEXX. The results are shown in Table 4. Further, by immunofluorescence, the diluted serum samples were used to detect PRRSV-infected Marc-145 cells, and the three samples were determined to be PRRSV antibody-positive samples, which was consistent with the method established by the present invention. The immunofluorescence results are shown in Figure 8.
表3Table 3
表4：使用爱德士公司商品化试剂盒对100份来自场地的猪血清样本进行ELISA检测结果Table 4: ELISA test results of 100 pig serum samples from the field using the commercial kit of IDEXX
序列表Sequence Listing
<110> 西北农林科技大学<110> Northwest Agriculture and Forestry University
<120> 一种猪血清样中病原抗体ELISA检测试剂盒<120> An ELISA kit for detecting pathogenic antibodies in pig serum samples
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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 2<400> 2
Asp Leu Pro Ala Lys Gly Thr Pro Val Asn Leu Ala Val Pro Arg GluAsp Leu Pro Ala Lys Gly Thr Pro Val Asn Leu Ala Val Pro Arg Glu
1 5 10 151 5 10 15
Glu Gln Leu Gly Gly Gly Ser Leu Val Ala Ser Leu Arg Pro Ile HisGlu Gln Leu Gly Gly Gly Ser Leu Val Ala Ser Leu Arg Pro Ile His
20 25 3020 25 30
Lys Gly Gly Gly Ser Gly Glu Ala Gln Met Leu Pro Glu Thr Val PheLys Gly Gly Gly Ser Gly Glu Ala Gln Met Leu Pro Glu Thr Val Phe
35 40 4535 40 45
Ser Thr Gly Arg Ile Glu Val Asp Cys Arg Gly Gly Gly Ser Leu ProSer Thr Gly Arg Ile Glu Val Asp Cys Arg Gly Gly Gly Ser Leu Pro
50 55 6050 55 60
His Ala Phe Ile Gly Asp Val Lys Gly Gly Gly Ser Asp Lys Thr AlaHis Ala Phe Ile Gly Asp Val Lys Gly Gly Gly Ser Asp Lys Thr Ala
65 70 75 8065 70 75 80
Tyr Phe Gln Leu Glu Gly Gly Gly Ser Asn Phe Leu Trp Met Leu SerTyr Phe Gln Leu Glu Gly Gly Gly Ser Asn Phe Leu Trp Met Leu Ser
85 90 9585 90 95
Arg Gly Gly Gly Ser Ile Met Glu Lys Ala Gly Gln Ala Ala Trp LysArg Gly Gly Gly Ser Ile Met Glu Lys Ala Gly Gln Ala Ala Trp Lys
100 105 110100 105 110
Gly Gly Gly Ser Ala Glu Thr Cys Lys Tyr Leu Ala Ser Arg Leu ProGly Gly Gly Ser Ala Glu Thr Cys Lys Tyr Leu Ala Ser Arg Leu Pro
115 120 125115 120 125
Met Leu Gly Gly Gly Ser Pro Thr Pro Gly Ser Arg Pro Lys Leu HisMet Leu Gly Gly Gly Ser Pro Thr Pro Gly Ser Arg Pro Lys Leu His
130 135 140130 135 140
Asp Phe Gln Gly Gly Gly Ser Asn Asp His Asp Glu Leu Gly Phe MetAsp Phe Gln Gly Gly Gly Ser Asn Asp His Asp Glu Leu Gly Phe Met
145 150 155 160145 150 155 160
Val Pro Pro Gly Leu Ser Ser Gly Gly Gly Ser Ala Leu Thr Thr SerVal Pro Pro Gly Leu Ser Ser Gly Gly Gly Ser Ala Leu Thr Thr Ser
165 170 175165 170 175
His Phe Leu Asp Thr Val Gly Leu Ala Gly Gly Gly Ser Val Leu AspHis Phe Leu Asp Thr Val Gly Leu Ala Gly Gly Gly Ser Val Leu Asp
180 185 190180 185 190
Gly Ser Ala Ala Thr Pro Leu Thr Arg Val Gly Gly Gly Ser Ala ProGly Ser Ala Ala Thr Pro Leu Thr Arg Val Gly Gly Gly Ser Ala Pro
195 200 205195 200 205
Gln Lys Val Leu Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val Gly GlyGln Lys Val Leu Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val Gly Gly
210 215 220210 215 220
Gly Ser Gly Arg Leu Leu Gly Leu Leu His Leu Leu Ile Phe Leu AsnGly Ser Gly Arg Leu Leu Gly Leu Leu His Leu Leu Ile Phe Leu Asn
225 230 235 240225 230 235 240
Cys Ala Phe Thr Phe Gly Tyr Met Gly Gly Gly Ser Thr Phe Val HisCys Ala Phe Thr Phe Gly Tyr Met Gly Gly Gly Ser Thr Phe Val His
245 250 255245 250 255
Phe Glu Ser Thr Asn Arg Val Ala Leu Thr Met Gly Gly Gly Ser LysPhe Glu Ser Thr Asn Arg Val Ala Leu Thr Met Gly Gly Gly Ser Lys
260 265 270260 265 270
Tyr Ile Leu Ala Pro Ala His His Val Glu Ser Gly Gly Gly Ser AlaTyr Ile Leu Ala Pro Ala His His Val Glu Ser Gly Gly Gly Ser Ala
275 280 285275 280 285
Gly Phe His Pro Ile Ala Ala Asn Asp Asn His Ala Phe Val Val ArgGly Phe His Pro Ile Ala Ala Asn Asp Asn His Ala Phe Val Val Arg
290 295 300290 295 300
Arg Pro Gly Ser Thr Thr Val Gly Gly Gly Ser Lys Gln Gly Val ValArg Pro Gly Ser Thr Thr Val Gly Gly Gly Ser Lys Gln Gly Val Val
305 310 315 320305 310 315 320
Asn Leu Val Lys Tyr Ala Lys Gly Gly Gly Ser Lys Gly Asn Gly GlnAsn Leu Val Lys Tyr Ala Lys Gly Gly Gly Ser Lys Gly Asn Gly Gln
325 330 335325 330 335
Pro Val Asn Gln Leu Gly Gly Gly Ser Lys Asn Pro Glu Lys Pro HisPro Val Asn Gln Leu Gly Gly Gly Ser Lys Asn Pro Glu Lys Pro His
340 345 350340 345 350
Phe Pro Leu Ala Thr Glu Gly Gly Gly Ser Thr Val Glu Phe Ser LeuPhe Pro Leu Ala Thr Glu Gly Gly Gly Ser Thr Val Glu Phe Ser Leu
355 360 365355 360 365
Pro Thr Gln His Thr Val Arg Leu Ile Arg Ala Thr Ala Ser Pro SerPro Thr Gln His Thr Val Arg Leu Ile Arg Ala Thr Ala Ser Pro Ser
370 375 380370 375 380
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu His His HisAla Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu His His
385 390 395 400385 390 395 400
His His HisHis His His
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