CN102023215B - Influenza virus antibody detection method based on eukaryotic cell agglutination reaction of recombined expression influenza virus hemagglutinin - Google Patents

本发明涉及生物技术领域，具体涉及一种基于重组表达流感病毒血凝素的真核细胞凝集反应的流感病毒抗体的检测方法。本发明检测方法是将流感病毒血凝素基因克隆后转染真核细胞，使真核细胞表面重组表达流感病毒血凝素蛋白，该蛋白与待检样品中的流感病毒血凝素抗体结合，发生凝集反应，根据是否发生凝集反应来检测待检样品中的流感病毒抗体。本发明检测方法快速、稳定，可以更广泛、快速地检测不同血清型的流感病毒抗体。The invention relates to the field of biotechnology, in particular to a method for detecting influenza virus antibodies based on eukaryotic cell agglutination of recombinantly expressed influenza virus hemagglutinin. The detection method of the present invention is to transfect eukaryotic cells after cloning the influenza virus hemagglutinin gene, so that the surface of the eukaryotic cells recombines and expresses the influenza virus hemagglutinin protein, and the protein binds to the influenza virus hemagglutinin antibody in the sample to be tested, The agglutination reaction occurs, and the influenza virus antibody in the sample to be tested is detected according to whether the agglutination reaction occurs. The detection method of the invention is rapid and stable, and can detect influenza virus antibodies of different serotypes more widely and rapidly.

一种基于重组表达流感病毒血凝素的真核细胞凝集反应的流感病毒抗体的检测方法A detection method for influenza virus antibody based on eukaryotic cell agglutination reaction of recombinantly expressed influenza virus hemagglutinin

技术领域 technical field

本发明涉及生物技术领域，具体涉及一种基于重组表达流感病毒血凝素的真核细胞凝集反应的流感病毒抗体的检测方法。The invention relates to the field of biotechnology, in particular to a method for detecting influenza virus antibodies based on eukaryotic cell agglutination of recombinantly expressed influenza virus hemagglutinin.

背景技术 Background technique

特定血清型流感病毒感染、感染后恢复、隐性感染、以及疫苗接种的人或动物，在其体液(如血清、支气管灌洗液、血浆、组织液等)中会出现流感病毒血凝素抗体，通过检测特定血清型流感病毒的抗体，可以判断、诊断或确诊该人或该动物是否感染(包括早期感染、隐性感染或感染后恢复等)该特定血清型的流感病毒、或接种过流感病毒疫苗、或评价其对特定血清型流感病毒的抵抗力等。Antibodies to influenza virus hemagglutinin will appear in body fluids (such as serum, bronchial lavage fluid, plasma, tissue fluid, etc.) of specific serotype influenza virus infection, recovery after infection, latent infection, and vaccination of humans or animals, By detecting antibodies to a specific serotype of influenza virus, it is possible to determine, diagnose or confirm whether the person or animal is infected (including early infection, latent infection or recovery after infection, etc.) with the specific serotype of influenza virus, or has been inoculated with influenza virus Vaccines, or evaluation of their resistance to specific serotypes of influenza viruses, etc.

目前检测待检样品中是否存在某一特定血清型的流感病毒抗体的方法，主要有血凝抑制实验(HAI)和中和实验，即将某一特定血清型的流感病毒先与待检样品混合，然后加入红细胞或敏感细胞中，判断待检样品是否能够抑制该病毒的血凝反应或细胞病变效应来进行判定，其缺点是需要使用具有感染性的病毒，安全性存在隐患。在涉及病毒的实验中，根据国家有关法规(国务院《病原微生物实验室生物安全管理条例》、卫生部《人间传染的病原微生物名录》、《人间传染的高致病性病原微生物实验室和实验活动生物安全审批管理办法》)及WHO的规定与建议，包括人H2N2以及部分禽流感病毒的操作要求在BSL-III(P3)实验室进行，因此，上述方法的开展对实验室条件要求很高，不适用于现场检测和规模检测。At present, the method for detecting whether there is a certain serotype of influenza virus antibody in the sample to be tested mainly includes hemagglutination inhibition test (HAI) and neutralization test, that is, the influenza virus of a certain serotype is first mixed with the sample to be tested, Then add red blood cells or sensitive cells to judge whether the sample to be tested can inhibit the hemagglutination reaction or cytopathic effect of the virus. The disadvantage is that infectious viruses need to be used, and there are hidden dangers in safety. In experiments involving viruses, according to the relevant national laws and regulations (State Council's "Regulations on the Biosafety Management of Pathogenic Microorganism Laboratories", the Ministry of Health's "List of Pathogenic Microorganisms Infected Between Humans", "Highly Pathogenic Microorganisms Infected Between Humans and Experimental Activities" "Biosafety Approval Management Measures") and WHO's regulations and recommendations, including the operation of human H2N2 and some avian influenza viruses are required to be carried out in BSL-III (P3) laboratories. Therefore, the development of the above methods requires high laboratory conditions. Not suitable for field testing and scale testing.

文献也有报道，利用表达流感病毒HA的假病毒颗粒来代替具有感染性的流感病毒进行HAI实验，但技术过于复杂。It has also been reported in the literature that pseudovirus particles expressing influenza virus HA are used to replace infectious influenza virus for HAI experiments, but the technique is too complicated.

除经典的HAI和中和实验外，也有用ELISA方法和乳胶凝集实验方法来进行检测的报道。其中，ELISA方法是利用重组表达和纯化的流感病毒HA来检测相应的抗体，这需要获保持抗原特异性的重组蛋白的表达与纯化，且由于流感病毒有众多亚型，即使同种亚型的血清交叉反应强度不一，对不同血清型的HA抗原都进行重组蛋白的表达与纯化，其工作量大、工艺要求高、批间稳定性差。乳胶凝集方法相对于现有的其它方法(HAI、中和实验以及ELISA方法)而言，反应时间短、适合现场检测，但仍存在一些不足。乳胶凝集实验方法是将重组表达和纯化的流感病毒HA蛋白偶联在惰性乳胶颗粒表面，利用凝集反应进行检测流感病毒相应的抗体，这同样需要重组表达和纯化的流感病毒HA，并需要获得保持抗原特异性的重组蛋白的表达与纯化，如需要检测不同血清型的流感病毒HA抗体，就需要对不同毒株的HA进行重组表达和纯化，与ELISA方法一样，存在工作量大、工艺要求高、批间稳定性差等问题。In addition to the classic HAI and neutralization experiments, ELISA methods and latex agglutination experiments are also used for detection reports. Among them, the ELISA method uses recombinantly expressed and purified influenza virus HA to detect corresponding antibodies, which requires the expression and purification of recombinant proteins that maintain antigen specificity, and because influenza viruses have many subtypes, even the same subtype The intensity of serum cross-reactivity varies, and the expression and purification of recombinant proteins are performed on HA antigens of different serotypes, which requires a large workload, high process requirements, and poor stability between batches. Compared with other existing methods (HAI, neutralization experiment and ELISA method), the latex agglutination method has short reaction time and is suitable for on-site detection, but there are still some shortcomings. The latex agglutination test method is to couple the recombinant expressed and purified influenza virus HA protein on the surface of inert latex particles, and use the agglutination reaction to detect the corresponding antibody of influenza virus, which also requires recombinant expressed and purified influenza virus HA, and needs to be maintained For the expression and purification of antigen-specific recombinant proteins, if it is necessary to detect HA antibodies of different serotypes of influenza virus, it is necessary to perform recombinant expression and purification of HA of different strains. Like the ELISA method, there are heavy workload and high process requirements. , poor inter-batch stability and other issues.

由于检测流感病毒抗体对人或该动物是否感染(包括早期感染、隐性感染或感染后恢复等)该特定血清型的流感病毒、或接种过流感病毒疫苗、或评价其对特定血清型流感病毒的抵抗力等有重要意义，而目前的HAI、中和实验、ELISA以及乳胶凝集等检测手段，或需要使用具有感染性的病毒，或需要特定的实验条件和设备，或工艺复杂，或检测耗时长等缺点，因此，一种简便、快速、工艺相对简单易控的流感病毒抗体检测方法具有广泛的应用价值。Due to the detection of influenza virus antibodies to humans or animals whether they are infected (including early infection, latent infection or recovery after infection, etc.) of the specific serotype of influenza virus, or have been vaccinated against influenza virus, or evaluated for specific serotype influenza virus However, the current detection methods such as HAI, neutralization experiment, ELISA and latex agglutination, or require the use of infectious viruses, or require specific experimental conditions and equipment, or the process is complicated, or the detection consumes Therefore, a simple, rapid, relatively simple and easy-to-control method for detecting influenza virus antibody has wide application value.

发明内容Contents of the invention

本发明的目的在于根据现有的检测流感病毒抗体技术中存在的工作量大、稳定性差、检测耗时长等问题，提供一种基于重组表达流感病毒血凝素的真核细胞凝集反应的流感病毒抗体的检测方法。The purpose of the present invention is to provide a kind of influenza virus based on the eukaryotic cell agglutination reaction of recombinantly expressed influenza virus hemagglutinin according to the problems of large workload, poor stability, and long detection time in the existing influenza virus antibody detection technology. Antibody detection method.

本发明目的通过以下技术方案予以实现：The object of the invention is achieved through the following technical solutions:

一种基于重组表达流感病毒血凝素(HA)的真核细胞凝集反应的流感病毒抗体检测方法，所述方法是将流感病毒血凝素基因克隆后转染真核细胞，使真核细胞表面重组表达流感病毒血凝素蛋白，以此为凝集反应基质，该蛋白与待检样品中的流感病毒血凝素抗体结合，发生凝集反应，根据是否发生凝集反应来检测待检样品中的流感病毒抗体。A kind of influenza virus antibody detection method based on the eukaryotic cell agglutination reaction of recombinant expression influenza virus hemagglutinin (HA), described method is transfected eukaryotic cell after the influenza virus hemagglutinin gene clone, makes eukaryotic cell surface Recombinant expression of influenza virus hemagglutinin protein, using this as the agglutination reaction matrix, the protein binds to the influenza virus hemagglutinin antibody in the sample to be tested, and agglutination occurs, and the influenza virus in the sample to be tested is detected according to whether the agglutination reaction occurs Antibody.

流感病毒包括甲型流感病毒、乙型流感病毒和丙型流感病毒，其中甲型流感病毒的HA可以分为16个亚型，同时，由于流感病毒HA的突变和进化，即使是同一亚型的HA，其交叉反应性的强弱不一。因此，检测某一特定血清型流感病毒抗体时，使用本发明的方法，将重组表达该特定血清型毒株的HA的真核细胞与待检样品进行凝集实验即可。上述的流感病毒HA，是指甲型流感病毒的H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16亚型，以及乙型流感病毒、丙型流感病毒的血凝素，可以根据检测目的的需要使用某一特定血清型的HA或其部分。Influenza viruses include influenza A virus, influenza B virus and influenza C virus. The HA of influenza A virus can be divided into 16 subtypes. At the same time, due to the mutation and evolution of influenza virus HA, even the same subtype HA, the strength of its cross-reactivity varies. Therefore, when detecting antibodies to a specific serotype of influenza virus, the method of the present invention is used to perform agglutination experiments on the eukaryotic cells recombinantly expressing the HA of the specific serotype strain and the sample to be tested. The above-mentioned influenza virus HA is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 subtypes of nail influenza virus, and influenza B Virus, hemagglutinin of influenza C virus, HA of a specific serotype or part thereof can be used according to the needs of the detection purpose.

如是进行较广泛血清型的流感病毒抗体检测，则可以根据所检测的目的血清型毒株的HA进行抗原性分析，选用抗原性保守或抗原交叉性强的HA蛋白的一部分进行重组表达真核细胞的构建，利用凝集反应对较广泛血清型的流感病毒抗体检测。上述的流感病毒HA，可以是某一流感病毒毒株HA或其中一部分，也可以是不同流感病毒毒株的HA或其中一部分或不同HA的拼接或混合，也可以是对流感病毒HA进行了突变和修饰，也可以是人工合成的天然不存在的序列，但其核心是重组表达产物具有流感病毒HA的抗原性。In the case of influenza virus antibody detection of a wider serotype, antigenicity analysis can be carried out according to the HA of the target serotype strain detected, and a part of the HA protein with conservative antigenicity or strong cross-cutting antigenicity can be selected for recombinant expression in eukaryotic cells The construction of the invention uses agglutination reaction to detect influenza virus antibodies of a wider range of serotypes. The above-mentioned influenza virus HA can be a certain influenza virus strain HA or a part thereof, can also be HA of a different influenza virus strain or a part thereof or a splicing or mixing of different HAs, or can be a mutation of the influenza virus HA And modification can also be artificially synthesized sequences that do not exist in nature, but the core is that the recombinant expression product has the antigenicity of influenza virus HA.

上述的重组表达流感病毒血凝素(HA)的真核细胞，是指将编码有流感病毒HA的基因片段导入真核细胞，在真核细胞表面重组表达流感病毒的HA。真核细胞可以是酵母、哺乳动物细胞、昆虫细胞，以及其它的真核细胞，重组表达的流感病毒HA位于细胞膜或细胞壁表面。其中，真核细胞优选哺乳动物细胞和昆虫细胞，其重组表达的流感病毒血凝素可以有更为接近病毒感染人或动物所获得翻译后修饰、空间构型和抗原性，其与流感病毒相应抗体的反应特异性更佳。进一步，优选哺乳动物细胞重组表达流感病毒HA。重组表达可以是瞬时表达，也可以是稳定表达。编码有流感病毒HA的基因片段可以是DNA，也可以是RNA，可以是单纯的含有启动子和编码序列的片段，也可以是质粒，也可以是病毒载体，其可以仅编码流感病毒HA，也可以同时或独立混合编码其它蛋白或多肽或肽段，其目的是将编码流感病毒HA的基因片段在真核细胞中获得重组表达。导入真核细胞的方式可以使用电击、脂质体转染、病毒介导等，目的是将编码流感病毒HA的基因片段进入真核细胞以得到重组表达。上述的重组表达流感病毒血凝素(HA)的真核细胞，其核心在于获得在细胞表面表达有流感病毒HA的真核细胞，其目的是利用所表达HA的抗原性，通过凝集实验，检测流感病毒抗体。在本发明的一个实施方案中，公布了重组表达流感病毒血凝素(HA)的真核细胞的方法。The aforementioned eukaryotic cells recombinantly expressing influenza virus hemagglutinin (HA) refers to introducing a gene segment encoding influenza virus HA into eukaryotic cells, and recombinantly expressing influenza virus HA on the surface of eukaryotic cells. The eukaryotic cells can be yeast, mammalian cells, insect cells, and other eukaryotic cells, and the recombinantly expressed influenza virus HA is located on the surface of the cell membrane or cell wall. Among them, eukaryotic cells are preferably mammalian cells and insect cells, and the recombinantly expressed influenza virus hemagglutinin can have post-translational modifications, spatial configuration and antigenicity that are closer to those obtained by virus-infected humans or animals, and it corresponds to influenza virus Antibody responses are more specific. Further, it is preferred that mammalian cells recombinantly express influenza virus HA. Recombinant expression can be transient expression or stable expression. The gene fragment encoding influenza virus HA can be DNA or RNA, it can be a simple fragment containing a promoter and coding sequence, it can also be a plasmid, or it can be a viral vector, which can only encode influenza virus HA, or The encoding of other proteins or polypeptides or peptides can be mixed simultaneously or independently, and the purpose is to obtain recombinant expression of the gene segment encoding influenza virus HA in eukaryotic cells. The way of introduction into eukaryotic cells can be electric shock, liposome transfection, virus-mediated, etc., and the purpose is to introduce the gene fragment encoding influenza virus HA into eukaryotic cells for recombinant expression. The core of the above-mentioned eukaryotic cells expressing influenza virus hemagglutinin (HA) is to obtain eukaryotic cells expressing influenza virus HA on the cell surface. The purpose is to use the antigenicity of the expressed HA to detect Antibodies to influenza virus. In one embodiment of the present invention, a method of recombinantly expressing influenza virus hemagglutinin (HA) in eukaryotic cells is disclosed.

上述的重组表达流感病毒血凝素(HA)的真核细胞，作为用于凝集反应的颗粒抗原基质，可以是天然的重组表达流感病毒血凝素的真核细胞，或是经过胰酶处理、机械分散、酶解分散、神经氨酸酶处理、固定剂固定、稳定剂处理、防腐剂处理的等工艺同时或选择性处理后的重组表达流感病毒血凝素的真核细胞，以更好的使“凝集反应基质”进行保存、防止自凝、增加凝集反应检测的灵敏性和稳定性。在本发明的一个实施方案中，公布了重组表达流感病毒血凝素(HA)的真核细胞的处理方法，以提高凝集反应检测的灵敏性和稳定性。The above-mentioned eukaryotic cells recombinantly expressing influenza virus hemagglutinin (HA), as the particle antigen substrate for agglutination reaction, can be natural recombinant expressing influenza virus hemagglutinin eukaryotic cells, or treated with trypsin, Recombinant eukaryotic cells expressing influenza virus hemagglutinin after simultaneous or selective treatment of mechanical dispersion, enzymatic dispersion, neuraminidase treatment, fixative fixation, stabilizer treatment, preservative treatment, etc., to better Preserve the "agglutination reaction matrix", prevent self-agglutination, and increase the sensitivity and stability of agglutination reaction detection. In one embodiment of the present invention, a treatment method of eukaryotic cells recombinantly expressing influenza virus hemagglutinin (HA) is disclosed to improve the sensitivity and stability of agglutination detection.

作为一种优选方案，所述流感病毒血凝素抗体为IgG、IgM、IgA、IgE、IgD中的一种或几种的混合物。As a preferred embodiment, the influenza virus hemagglutinin antibody is one or a mixture of IgG, IgM, IgA, IgE, and IgD.

作为一种优选方案，所述真核细胞为酵母、哺乳动物细胞、昆虫细胞或其它真核细胞，重组表达的流感病毒血凝素位于细胞膜或细胞壁表面。As a preferred solution, the eukaryotic cells are yeast, mammalian cells, insect cells or other eukaryotic cells, and the recombinantly expressed influenza virus hemagglutinin is located on the surface of the cell membrane or cell wall.

作为一种优选方案，所述待检样品为人或动物的体液，如血清、支气管灌洗液、血浆或组织液。As a preferred solution, the sample to be tested is human or animal body fluid, such as serum, bronchial lavage fluid, plasma or tissue fluid.

与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

本发明是以重组表达流感病毒HA的细胞为载体，利用凝集反应，检测待检样品中是否存在相应流感病毒的抗体。利用RT-PCR克隆特定血清型流感病毒的HA基因或其片段，构建真核表达载体，转染真核细胞，细胞表面会存在重组表达的HA，经醛化固定后，该细胞即成为表面含有HA抗原的致敏颗粒，与待检样品混合后，如待检样品中含有流感病毒的抗体，即会在数分钟发生凝集反应。该方法不涉及具有感染性的病毒颗粒；相比ELISA、HAI和中和实验所需的数小时至数天，重组表达HA的细胞凝集反应仅需数分钟，反应时间快速；利用通用引物和载体可对不同血清型的HA基因进行克隆与转染表达，即可得到不同HA抗原的致敏细胞颗粒，可以更加广泛地对不同血清型流感病毒进行检测。The invention uses the cell expressing recombinant influenza virus HA as a carrier, utilizes agglutination reaction, and detects whether the corresponding influenza virus antibody exists in the sample to be tested. Use RT-PCR to clone the HA gene or its fragment of a specific serotype influenza virus, construct a eukaryotic expression vector, and transfect eukaryotic cells. There will be recombinantly expressed HA on the cell surface. After the sensitizing particles of HA antigen are mixed with the sample to be tested, if the sample to be tested contains antibodies to influenza virus, agglutination will occur within a few minutes. The method does not involve infectious virus particles; the agglutination reaction of cells expressing recombinant HA takes only a few minutes, compared to the hours to days required for ELISA, HAI, and neutralization experiments; the reaction time is fast; Utilizes universal primers and vectors The HA genes of different serotypes can be cloned, transfected and expressed, and the sensitized cell particles of different HA antigens can be obtained, which can more widely detect different serotypes of influenza viruses.

具体实施方式Detailed ways

以下结合实施例来进一步解释本发明，但实施例并不对本发明做任何形式的限定。The present invention is further explained below in conjunction with the examples, but the examples do not limit the present invention in any form.

实施例1重组表达流感病毒血凝素(HA)的真核细胞的制备The preparation of the eukaryotic cell of embodiment 1 recombinant expression influenza virus hemagglutinin (HA)

本实施例以一株H5亚型的毒株A/Chicken/Guangdong/1/2005(H5N1)为例，进行重组表达流感病毒血凝素HA真核表达细胞的制备。A/Chicken/Guangdong/1/2005(H5N1)是2005年在广东省的鸡中分离得到的一株H5N1亚型流感病毒，其HA的基因序列可在公共数据库GenBank上获得，其序列号为EU874899.2。将该株病毒增殖后使用Viral RNA Miniprep Kit提取病毒RNA，用Uni-12引物(5’-AGCAAAAGCAGG-3’)和SuperScript III或M-MLV逆转录酶进行逆转录，得到病毒cDNA。根据该毒株HA的基因序列，设计一对用于HA全长基因克隆的引物，其引物序列具体如下：上游引物H5HA-F，序列为5’-GCAAAGCTTACCATGGAGAAAATACTTCTTC-3’，从5’端依次为3个保护性碱基、Hind III酶切位点、KOZAK序列、起始密码子和配对区；下游引物H5HA-R，序列为5’-CTCGGATCCTTAAATGCAAATTCTGCATTGTAAG-3’，从5’端依次为3个保护性碱基、BamH I酶切位点、终止密码子和配对区。利用这对引物，用高保真Taq酶对病毒cDNA进行PCR扩增，扩增HA片段。PCR产物经琼脂糖凝胶电泳回收后，与表达载体pcDNA3分别进行使用Hind III和BamH I双酶切，再次电泳回收后利用T4DNA连接酶将酶切回收后的HA片段与载体进行连接，转化大肠杆菌DH5a感受态细胞，涂布Amp抗性平板。菌落经PCR后，提取质粒酶切鉴定和DNA测序，获得含有HA基因片段的真核表达质粒，即pcDNA-H5HA。In this example, an H5 subtype strain A/Chicken/Guangdong/1/2005 (H5N1) was taken as an example to prepare eukaryotic cells expressing recombinant influenza virus hemagglutinin HA. A/Chicken/Guangdong/1/2005 (H5N1) is an H5N1 subtype influenza virus isolated from chickens in Guangdong Province in 2005. The gene sequence of its HA can be obtained on the public database GenBank, and its sequence number is EU874899 .2. Viral RNA was extracted using Viral RNA Miniprep Kit after the virus was propagated, and reverse transcription was performed with Uni-12 primer (5'-AGCAAAAGCAGG-3') and SuperScript III or M-MLV reverse transcriptase to obtain viral cDNA. According to the gene sequence of the strain HA, a pair of primers for HA full-length gene cloning was designed. 3 protective bases, Hind III restriction site, KOZAK sequence, start codon and pairing region; the downstream primer H5HA-R, the sequence is 5'-CTCGGATCCTTAAATGCAAATTCTGCATTGTAAG-3', and there are 3 protections from the 5' end sex base, BamH I restriction site, stop codon and pairing region. Utilize this pair of primers, carry out PCR amplification to virus cDNA with high-fidelity Taq enzyme, amplify HA fragment. After the PCR product was recovered by agarose gel electrophoresis, it was digested with the expression vector pcDNA3 using Hind III and BamH I respectively. After the recovery by electrophoresis again, the HA fragment recovered by enzyme digestion was ligated with the carrier by T4DNA ligase, and transformed into large intestine Bacillus DH5a competent cells, coated with Amp resistance plates. After the colonies were subjected to PCR, the plasmids were extracted and identified by enzyme digestion and DNA sequencing to obtain a eukaryotic expression plasmid containing HA gene fragments, ie pcDNA-H5HA.

含有pcDNA-H5HA质粒的细菌，经含有50μg/ml的氨苄青霉素(Amp)的LB培养过夜后，用高纯度质粒提取试剂盒提取pcDNA-H5HA质粒。根据Lipofectamine 2000试剂盒说明，将3μg pcDNA-H5HA质粒和10μl Lipofectamine2000转染复合物，转染一个35mm直径培养皿含有生长密度约为80％-90％融合率的人胚肾HEK-239细胞，转染后6h去除转染复合物，更换新鲜的完全培养基(含有10％胎牛血清的DMEM培养基)。The bacteria containing the pcDNA-H5HA plasmid were cultured overnight in LB containing 50 μg/ml ampicillin (Amp), and the pcDNA-H5HA plasmid was extracted with a high-purity plasmid extraction kit. According to the instructions of the Lipofectamine 2000 kit, 3 μg pcDNA-H5HA plasmid and 10 μl Lipofectamine2000 transfection complex were transfected into a 35 mm diameter culture dish containing human embryonic kidney HEK-239 cells with a growth density of about 80%-90% fusion rate, and transfected 6h after transfection, the transfection complex was removed, and fresh complete medium (DMEM medium containing 10% fetal bovine serum) was replaced.

转染后，HEK-239细胞即可瞬时重组表达HA，即得到瞬时重组表达流感病毒HA的细胞。After transfection, HEK-239 cells can transiently recombinantly express HA, that is, cells transiently recombinantly expressing influenza virus HA can be obtained.

为进一步优化，可以筛选获得稳定重组表达HA的细胞：在转染72h后将转染细胞吹打分散，将细胞悬液的1/20接种一个新的35mm直径培养皿中，补充完全培养基至3ml，经过夜贴壁后，补加G418至浓度为600μg/ml。此后每3至5天的更换一次含有600μg/ml G418的完全培养基，约3周后可见筛选出现的抗性细胞集落。将抗性细胞集落经胰酶消化后，按10-20个细胞/ml接种96孔板培养，加入含有600μg/ml G418的完全培养基培养至长至单层后，进行传代，获得用于检测的备份培养96孔板。将检测用的96孔板的细胞用4％多聚甲醛室温固定5分钟，经PBS漂洗后，加入抗H5亚型HA的抗体为一抗、Cy3荧光标记的二抗进行免疫荧光检测，选择荧光阳性信号强、荧光阳性细胞比例高的孔，对细胞继续重复的克隆化和免疫荧光，至阳性细胞比例接近100％，即可得到稳定重组表达该毒株HA的细胞。For further optimization, cells with stable recombinant expression of HA can be screened: After 72 hours of transfection, the transfected cells were blown and dispersed, and 1/20 of the cell suspension was inoculated into a new 35mm diameter culture dish, and the complete medium was supplemented to 3ml , after overnight adherence, G418 was added to a concentration of 600 μg/ml. Thereafter, the complete medium containing 600 μg/ml G418 was replaced every 3 to 5 days, and the resistant cell colonies that appeared after screening could be seen after about 3 weeks. Digest resistant cell colonies with trypsin, inoculate 10-20 cells/ml in a 96-well plate for culture, add complete medium containing 600 μg/ml G418 and culture until it grows to a monolayer, then pass passage to obtain Back up cultures in 96-well plates. Fix the cells in the 96-well plate for detection with 4% paraformaldehyde at room temperature for 5 minutes, rinse with PBS, add anti-H5 subtype HA antibody as the primary antibody, and Cy3 fluorescently labeled secondary antibody for immunofluorescence detection. For the wells with strong positive signal and high proportion of fluorescent positive cells, repeat cloning and immunofluorescence on the cells until the proportion of positive cells is close to 100%, then cells stably recombinantly expressing the strain HA can be obtained.

实施例2重组表达流感病毒血凝素(HA)的真核细胞的处理Example 2 Recombinant treatment of eukaryotic cells expressing influenza virus hemagglutinin (HA)

瞬时或稳定重组表达该毒株HA的细胞，经培养后，用低浓度胰酶(0.05％)温和消化细胞约5分钟，加入含有胎牛血清的培养基终止反应，将细胞悬液进行离心，去除上清后用含有2％BSA的PBS进行重悬，获得胰酶处理后的细胞悬液。Transiently or stably recombinantly expressing the cells of the strain HA, after culturing, gently digest the cells with low concentration of trypsin (0.05%) for about 5 minutes, add a medium containing fetal bovine serum to terminate the reaction, and centrifuge the cell suspension. After removing the supernatant, resuspend with PBS containing 2% BSA to obtain the cell suspension after trypsin treatment.

为防止HA可能引起的细胞自凝集，在胰酶处理后的细胞悬液中加入神经氨酸酶(又称神经氨酸苷酶，Neuraminidase)进行处理，消除重组表达的HA与细胞自身受体的作用，避免细胞自身凝集。即，将细胞悬液经离心后，用50mM柠檬酸钠(pH 6.0)缓冲液，将细胞按10⁷个/ml进行重悬，每ml细胞悬液中加入200单位的神经氨酸酶，于37℃条件下处理30分钟后离心，去除上清，用等体积的PBS重悬、离心进行洗涤2次，再次加入等体积的PBS。In order to prevent cell self-agglutination that may be caused by HA, neuraminidase (also known as neuraminidase, Neuraminidase) was added to the cell suspension after trypsin treatment to eliminate the interaction between the recombinantly expressed HA and the cell's own receptors. function to prevent cell agglutination. That is, after the cell suspension was centrifuged, 50 mM sodium citrate (pH 6.0) buffer was used to resuspend the cells at 10 ⁷ cells/ml, and 200 units of neuraminidase was added to each ml of the cell suspension. After being treated at 37°C for 30 minutes, centrifuge, remove the supernatant, resuspend with an equal volume of PBS, wash by centrifugation twice, and add an equal volume of PBS again.

上述经胰酶和神经氨酸酶处理的细胞悬液，加入等体积的10％的冷甲醛，于4℃固定2小时。经离心、PBS重悬的方式进行3次洗涤。最后将细胞沉淀，按10⁷个/ml的浓度重悬于含有0.2％尼泊尔金酯、2％BSA、20％甘油的PBS缓冲液中，使其能够稳定地长时间保存，即得到了处理后的重组表达HA抗原的真核细胞。The above-mentioned cell suspension treated with trypsin and neuraminidase was added with an equal volume of 10% cold formaldehyde, and fixed at 4° C. for 2 hours. Wash 3 times by centrifugation and resuspension in PBS. Finally, the cell pellet was resuspended at a concentration of 10 ⁷ cells/ml in PBS buffer containing 0.2% Nepalese gold ester, 2% BSA, and 20% glycerol, so that it could be stored stably for a long time, that is, after treatment Recombinant eukaryotic cells expressing HA antigen.

实施例3使用重组表达流感病毒血凝素的真核细胞进行凝集反应检测流感病毒抗体Example 3 Using eukaryotic cells recombinantly expressing influenza virus hemagglutinin to perform agglutination reaction to detect influenza virus antibodies

将实施例2中的处理后的重组表达HA抗原的真核细胞悬液1滴(约50μl)加到洁净的载玻片上，然后加入50μl的待检样品(血清、支气管灌洗液、血浆、组织液等)，前后左右轻轻晃动载玻片或使用微量移液器吸头将其混匀，室温下1-3分钟观察是否出现细胞凝集。Add 1 drop (approximately 50 μl) of the treated recombinant eukaryotic cell suspension expressing the HA antigen in Example 2 to a clean glass slide, and then add 50 μl of the sample to be tested (serum, bronchial lavage fluid, plasma, tissue fluid, etc.), gently shake the slide back and forth or use a micropipette tip to mix it, and observe whether there is cell agglutination at room temperature for 1-3 minutes.

设立阳性血清和阴性血清对照，如对照样品符合预期结果，待检样品发生未凝集时，说明待检样品中不含有与该毒株HA具有反应性的流感病毒HA抗体或抗体浓度低于该方法所能达到的检测浓度。Set up positive serum and negative serum controls. If the control sample meets the expected results and the sample to be tested does not agglutinate, it means that the sample to be tested does not contain influenza virus HA antibodies reactive with the strain HA or the antibody concentration is lower than that of the method. Achievable detection concentration.

待检样品发生凝集时，说明待检样品中含有与该毒株HA具有反应性的流感病毒HA抗体。此时可以进一步进行相对定量分析，即，将待检样品1进行连续的倍比稀释，然后分别与实施例2中的处理后的重组表达HA抗原的真核细胞悬液进行凝集反应，得到所能发生凝集的最高稀释度，设为d1。同样方法可得到待检样品2的凝集反应阳性最高稀释度d2。以此横向比较不同待检样品中流感病毒HA抗体的浓度差异和相对比例。When agglutination occurs in the sample to be tested, it indicates that the sample to be tested contains influenza virus HA antibodies reactive with the strain HA. At this time, relative quantitative analysis can be further carried out, that is, the sample 1 to be tested is serially diluted, and then agglutinated with the treated recombinant eukaryotic cell suspension expressing the HA antigen in Example 2 to obtain the obtained The highest dilution that can cause agglutination is set as d1. The highest agglutination reaction positive dilution d2 of the sample 2 to be tested can be obtained by the same method. In this way, the concentration difference and relative ratio of influenza virus HA antibody in different samples to be tested were compared horizontally.

一种基于重组表达流感病毒血凝素的真核细胞凝集反应的流感病毒抗体的检测方法序列表A detection method for influenza virus antibody based on eukaryotic cell agglutination reaction of recombinantly expressed influenza virus hemagglutinin

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