CN102023213B - Fluorescence micro cell agglutination method for detecting influenza virus antibody - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a fluorescent micro-cell agglutination method for detecting influenza virus antibodies. The method of the invention is to clone the influenza virus hemagglutinin gene and the neuraminidase gene and then transfect the eukaryotic cells, so that the surface of the eukaryotic cells recombines and expresses the influenza virus hemagglutinin and neuraminidase proteins, and performs fluorescence on the recombinantly expressed cells. Marking, the recombinantly expressed hemagglutinin and neuraminidase proteins on the surface of the cell combine with the influenza virus hemagglutinin antibody and neuraminidase antibody of the sample to be tested, and agglutination occurs, and the fluorescent microscope is photographed, and according to whether the fluorescent area Increase to determine whether there is influenza virus antibody in the sample to be tested. The detection method of the invention uses fluorescently labeled cells, reduces the amount of cells used, can perform high-throughput detection, and can realize rapid detection, early detection, on-site detection and evaluation of influenza virus antibodies.

Description

A fluorescent micro-cell agglutination method for detecting influenza virus antibody

technical field

The invention relates to the field of biotechnology, in particular to a fluorescent micro-cell agglutination method for detecting influenza virus antibodies.

Background technique

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.

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. 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.

A further disadvantage of the above method is that only antibodies to influenza virus HA can be detected. There are mainly two kinds of glycoprotein spikes with strong antigenicity on the surface of influenza virus, namely hemagglutinin NA and neuraminidase NA. When influenza virus infection or recessive infection or vaccination, human or animal body will target Both HA and NA produce antibodies, which is exactly the case. According to the antigenicity of HA, influenza A virus is divided into 16 subtypes such as H1-H16, and the antigenicity of NA is divided into 9 subtypes such as N1-N9. In influenza A virus, many subtypes are formed according to different combinations of HA and NA subtypes, such as H5N1 (that is, the combination of H5 subtype and N1 subtype). Therefore, in the detection of influenza virus antibodies, in addition to the detection of HA serotype, the detection of NA serotype is also required. However, the current detection of NA serotype antibodies requires an additional method of neuraminidase enzyme activity inhibition.

In addition to the classic HAI and neutralization experiments, ELISA methods and latex agglutination experiments are also used for detection reports. The advantage of ELISA and latex agglutination is that antibodies to HA and NA can be detected. Among them, the ELISA method uses recombinantly expressed and purified influenza virus HA and/or NA 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 if the same The serum cross-reactivity of different subtypes is different, and the expression and purification of recombinant proteins for different serotypes of HA and NA antigens requires a large workload, high process requirements, and poor batch-to-batch stability. 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 and/or NA protein on the surface of inert latex particles, and use the agglutination reaction to detect the corresponding antibody of influenza virus, which also requires the recombinant expressed and purified influenza virus HA and NA, and need to obtain the expression and purification of recombinant proteins that maintain antigen specificity. If it is necessary to detect influenza virus HA and NA antibodies of different serotypes, it is necessary to perform recombinant expression and purification of HA and NA of different strains, and the ELISA method Similarly, there are problems such as heavy workload, high process requirements, and poor stability between batches.

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 process that can simultaneously detect influenza virus HA and NA antibody detection methods has a wide range of application values.

Using eukaryotic cells recombinantly expressing influenza virus hemagglutinin (HA) and neuraminidase (NA) as the agglutination reaction matrix, using agglutination reaction to detect whether there are antibodies to the corresponding influenza virus in the sample to be tested, this method is relatively In terms of the above traditional methods, it is safer and faster to detect influenza virus antibodies. However, due to the disadvantages of limited throughput and large cell consumption in this method, a method suitable for high-throughput detection, with less cell consumption, simple and fast, relatively simple and easy-to-control process, and capable of simultaneously detecting influenza virus HA and NA antibody detection method.

Contents of the invention

The object of the present invention is to provide a simple, fast, less cell consumption, and other disadvantages in the prior art for detecting influenza virus antibodies, such as large cell consumption, complex process, long detection time, and limited throughput. A method for detecting antibodies to influenza virus suitable for high-throughput detection.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

The invention clones the influenza virus hemagglutinin gene and neuraminidase gene and then transfects eukaryotic cells, so that the surface of the eukaryotic cells recombines and expresses the influenza virus hemagglutinin and neuraminidase proteins, and performs fluorescent labeling on the recombinant expression cells , the recombinantly expressed hemagglutinin and neuraminidase proteins on the cell surface combine with the influenza virus hemagglutinin antibody and neuraminidase antibody of the sample to be tested, and an agglutination reaction occurs. Fluorescent detection and analysis, according to whether the fluorescent area increases Determine whether there is influenza virus antibody in the sample to be tested.

As a preferred solution, the fluorescent marker is fluorescein, fluorescent protein or other substances capable of emitting fluorescence; the equipment used for the fluorescence detection and analysis is a fluorescence microscope, a fluorescence photometer or a multifunctional microplate detector.

Use RT-PCR to clone the HA and NA genes or fragments of specific serotype influenza viruses, construct eukaryotic expression vectors, and transfect eukaryotic cells. There will be recombinantly expressed HA and NA on the cell surface. That is to say, it becomes a sensitized particle containing HA and NA antigens on the surface. During transfection, the cells can be simultaneously transfected with proteins or polypeptides or mutants that encode fluorescent proteins (green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, etc.) that can emit fluorescence under excitation light. ) nucleic acid, the cell can be fluorescently labeled. Fluorescent labeling can also use fluorescent substances, such as CFSE, SYTO, Cy3, Cy5, FITC, etc., to label cells. After the fluorescently labeled cells are mixed with the sample to be tested in a micro-agglutination plate, if the sample to be tested contains antibodies to influenza virus, agglutination will occur within a few minutes, and it can be clearly observed and judged whether agglutination occurs under a fluorescent microscope . At the same time, it is also possible to use image analysis software such as Image Pro Plus to analyze the fluorescence area of each well after taking pictures and imaging, and judge whether agglutination occurs by using the cells that are not easy to precipitate after agglutination and thus have a large fluorescence immunity.

Specifically, the technical scheme adopted in the present invention is as follows:

Eukaryotic cells that recombinantly express influenza virus hemagglutinin (HA), or eukaryotic cells that recombinantly express neuraminidase (NA), or eukaryotic cells that express both HA and NA, or recombinantly express HA and NA The eukaryotic cell mixture is labeled with a fluorescent substance. After the fluorescently labeled cells are mixed with the sample to be tested in a micro-agglutination plate, if the sample to be tested contains antibodies to influenza virus, an agglutination reaction will occur within a few minutes. Under a fluorescent microscope, it can be clearly observed and judged whether agglutination occurs. At the same time, it is also possible to use image analysis software such as Image Pro Plus to analyze the fluorescence area of each well after taking pictures and imaging, and judge whether agglutination occurs by using the cells that are not easy to precipitate after agglutination and thus have a large fluorescence immunity. If agglutination occurs, it indicates whether there is an antibody to the corresponding influenza virus in the sample to be tested.

As a preferred solution, the recombinant expression is transient expression or stable expression.

Influenza viruses include influenza A virus, influenza B virus and influenza C virus, wherein the HA of influenza A virus can be divided into 16 subtypes, and the NA can be divided into 9 subtypes. At the same time, due to the influenza virus HA and NA Even for the same subtype of HA and NA, the strength of 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. The above-mentioned influenza virus NA is the neuraminidase of nail type influenza virus N1, N2, N3, N4, N5, N6, N7, N8, N9 subtypes, influenza B virus and influenza C virus. The above-mentioned recombinantly expressed influenza virus HA and NA may be any combination of the above-mentioned different HA and NA.

As a preferred embodiment, the influenza virus hemagglutinin antibody and neuraminidase antibody are one or a mixture of IgG, IgM, IgA, IgE, and IgD.

In the case of influenza virus antibody detection of a wider serotype, antigenicity analysis can be carried out according to the HA and NA of the target serotype strain detected, and a part of HA and NA proteins with conservative antigenicity or strong antigenic crossability can be selected for recombination The construction of expressing eukaryotic cells, using agglutination reaction to detect influenza virus antibodies of a wider range of serotypes. The above-mentioned influenza virus HA and NA can be a certain influenza virus strain HA and NA or a part thereof, or HA and NA of different influenza virus strains or a part thereof or a splicing or mixing of different HA and NA. It is the mutation and modification of influenza virus HA and NA, or it can be artificially synthesized sequences that do not exist in nature, but its core is that the recombinant expression product has the antigenicity of influenza virus HA and NA.

The aforementioned eukaryotic cells recombinantly expressing influenza virus HA and NA refer to introducing gene fragments encoding influenza virus HA and NA into eukaryotic cells, and recombinantly expressing influenza virus HA and NA 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 and NA are 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 and NA. Recombinant expression can be transient expression or stable expression. The gene fragment encoding influenza virus HA and NA 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 and NA can also encode other proteins or polypeptides or peptides simultaneously or independently, the purpose of which is to obtain recombinant expression of the gene fragments encoding influenza virus HA and NA 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 gene fragments encoding influenza virus HA and NA into eukaryotic cells for recombinant expression. The core of the above-mentioned eukaryotic cells expressing HA and NA recombinantly is to obtain eukaryotic cells expressing influenza virus HA and NA on the cell surface, and the cells are fluorescently labeled with fluorescent substances. The influenza virus antibody can be detected by the agglutination test with a small amount of cells. In an embodiment of the present invention, methods for fluorescently labeled recombinant eukaryotic cells expressing influenza virus HA and NA are disclosed.

The eukaryotic cells used in the detection method of the present invention 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. The purpose of using eukaryotic cells for recombinant expression is to live influenza virus hemagglutinin and neuraminidase proteins or partial peptides with post-translational modifications (such as glycosylation, etc.) to better interact with influenza virus hemagglutination Recognition, binding and agglutination of the protein antibody and neuraminidase antibody or one of the two to obtain better affinity and specificity.

Compared with the prior art, the present invention has the following beneficial effects:

The fluorescent micro-cell agglutination method of the present invention for detecting influenza virus antibodies does not involve infectious virus particles; compared with the hours to days required for ELISA, HAI and neutralization experiments, fluorescently labeled recombinant expressing HA and NA cells agglutinate The reaction time is less than 60 minutes, and the reaction time is fast; HA and NA genes of different serotypes can be cloned, transfected and expressed by using universal primers and vectors, and sensitized cell particles of different HA and NA antigens can be obtained, which can be more extensively targeted Different serotypes of influenza virus antibodies are detected; compared with the non-fluorescence-labeled recombinant expression of HA and NA cell agglutination reaction method, the present invention is suitable for high-throughput detection, and the amount of cells is less (only 1/100 of cells not labeled with fluorescence) to 1/10).

Detailed ways

The present invention is further explained below in conjunction with the examples, but the examples do not limit the present invention in any form.

Embodiment 1 The method for the recombinant expression of influenza virus HA and NA eukaryotic cells labeled with green fluorescent protein

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 sequences of its HA and NA can be obtained on the public database GenBank, and the sequence of HA The number is EU874899.2, and the serial number of NA is EU874900.2. Viral RNA was extracted using Viral RNA Miniprep Kit after the strain 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.

According to the gene sequence of the strain NA, a pair of primers for NA full-length gene cloning was designed. 3 protective bases, Hind III restriction site, KOZAK sequence, start codon and pairing region; downstream primer N1NA-R, the sequence is: 5'-CTCGGATCCCTACTTGTCAATGGTGAATG-3', 3 in order from the 5' end Protective bases, BamH I restriction sites, stop codons and pairing regions. Utilize this pair of primers, carry out PCR amplification to virus cDNA with high-fidelity Taq enzyme, amplify NA fragment.

After the PCR product was recovered by agarose gel electrophoresis, the HA gene fragment was recovered, and the expression vector pcDNA3 was digested with Hind III and BamH I respectively. connect. The NA fragment and the green fluorescent expression vector were digested with Hind III and BamH I, and recovered by electrophoresis for connection. There is a ribosome entry site sequence (IRES) between the green fluorescent protein encoded by the vector and the multiple cloning site, and the NA gene After the fragment is inserted into the promoter of the vector and before the IRES, an mRNA encoding NA, IRES and green fluorescent protein can be obtained when the promoter initiates transcription, and finally translated to obtain separate NA and green fluorescent protein.

The ligation products were respectively transformed into Escherichia coli DH5a competent cells, and coated with Amp resistance plates. After the colonies were subjected to PCR, the plasmids were extracted for identification and DNA sequencing to obtain the eukaryotic expression plasmid containing the HA gene fragment, namely pcDNA-H5HA, and the eukaryotic expression plasmid containing the HA gene fragment, namely pIRES-EGFP-N1NA. Bacteria containing pcDNA-H5HA plasmid and pIRES-EGFP-N1NA plasmid were respectively cultured in LB containing 50 μg/ml ampicillin (Amp) overnight, and the high-purity plasmid extraction kit was used to extract pcDNA-H5HA plasmid and pIRES-EGFP- N1NA plasmid for subsequent transfection of cells and recombinant expression experiments.

According to the instructions of the Lipofectamine 2000 kit, transfect a 35mm diameter petri dish with a growth density of about 80%-90% fusion rate with 2.5 μg pcDNA-H5HA plasmid, 0.5 μg pIRES-EGFP-N1NA plasmid and 10 μl Lipofectamine 2000 transfection complex The human embryonic kidney HEK-239 cells were transfected, and the transfection complex was removed 6 hours after transfection, and fresh complete medium (DMEM medium containing 10% fetal bovine serum) was replaced.

After transfection, HEK-239 cells can transiently recombinantly express HA, NA and green fluorescent protein, that is, cells that recombinantly express influenza virus HA and NA labeled with transient green fluorescent protein can be obtained.

For further optimization, cells that stably express recombinant HA and NA labeled with green fluorescent protein can be screened: 72 hours after transfection, the transfected cells were dispersed by pipetting, and 1/20 of the cell suspension was inoculated into a new 35mm diameter culture dish , supplemented with complete medium to 3ml, after overnight attachment, added G418 to a concentration of 600μg/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 Cy3 fluorescently labeled anti-H5 subtype HA antibody for immunofluorescence detection, and observe the green fluorescence at the same time, select red and For the wells with strong green fluorescence positive signal and high proportion of red and green fluorescence positive cells, repeat cloning and immunofluorescence of the cells until the proportion of positive cells is close to 100%, then stable green fluorescent protein marker recombinant expression of the strain HA can be obtained and NA cells.

Label the cells with green fluorescent protein transiently or stably recombinantly expressing HA and NA of the strain. After adherent or suspension culture, the cells are dispersed by blowing with PBS containing 2% BSA, the cell suspension is centrifuged, and the supernatant is removed. Resuspend with PBS containing 2% BSA at a cell density of 10 ⁵ cells/ml. Remove the supernatant, resuspend with an equal volume of PBS, wash by centrifugation twice, add an equal volume of PBS again, add an equal volume of 10% cold formaldehyde, and fix at 4°C for 2 hours. Wash 3 times by centrifugation and resuspension in PBS. Finally, the cell pellet was resuspended in PBS buffer containing 0.2% Nepalese gold ester, 2% BSA, 20% glycerol, and 0.5% heparin at a concentration of 10 ⁵ cells/ml, so that it could be stored stably for a long time, that is, The treated eukaryotic cells labeled with green fluorescent protein and recombinantly expressing HA and NA antigens were obtained.

25 μl of the green fluorescent protein-labeled recombinant eukaryotic cell suspension expressing HA and NA antigens and 25 μl of the sample to be tested were mixed in a microagglutination plate, left at room temperature for 60 minutes, and then observed under a fluorescent microscope. If no agglutination occurs, the green fluorescent cells will settle at the bottom of the agglutination plate, and the fluorescent cells can be seen gathered in the center under the microscope; if agglutination occurs, the green fluorescent cells will be suspended in the liquid in flocculent form, and the fluorescent cells can be seen under the microscope Scattered across the field of view, clumping from cell to cell. Utilizing this feature, it is also possible to perform imaging with a fluorescence microscope, and use image analysis software to realize whether the software judges agglutination or not by setting up the fluorescent light-emitting area. When agglutination occurs in the sample to be tested, it indicates that the sample to be tested contains influenza virus HA and/or NA antibodies reactive with the strain HA and/or NA.

Embodiment 2 Fluorescein CFSE (carboxyfluorescein diacetate succinimidyl ester)-labeled method for eukaryotic cells expressing influenza virus HA and NA

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 sequences of its HA and NA can be obtained on the public database GenBank, and the sequence of HA The number is EU874899.2, and the serial number of NA is EU874900.2. 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.

According to the gene sequence of the strain NA, a pair of primers for NA full-length gene cloning was designed. 3 protective bases, Hind III restriction site, KOZAK sequence, start codon and pairing region; downstream primer N1NA-R, the sequence is: 5'-CTCGGATCCCTACTTGTCAATGGTGAATG-3', 3 in order from the 5' end Protective bases, BamH I restriction sites, stop codons and pairing regions. Utilize this pair of primers, carry out PCR amplification to virus cDNA with high-fidelity Taq enzyme, amplify NA fragment.

After the HA and NA gene fragments were recovered by agarose gel electrophoresis, the PCR products were digested with the expression vector pcDNA3 respectively using Hind III and BamH I. Carriers were ligated, NA fragments were ligated with the carrier, Escherichia coli DH5a competent cells were respectively transformed, and Amp resistance plates were coated. After the colonies were subjected to PCR, the plasmids were extracted for identification and DNA sequencing to obtain the eukaryotic expression plasmid containing the HA gene fragment, namely pcDNA-H5HA, and the eukaryotic expression plasmid containing the HA gene fragment, namely pcDNA-N1NA. Bacteria containing the pcDNA-H5HA plasmid and the pcDNA-N1NA plasmid were cultured overnight in LB containing 50 μg/ml ampicillin (Amp) respectively, and the pcDNA-H5HA plasmid and the pcDNA-N1NA plasmid were extracted with a high-purity plasmid extraction kit. In subsequent transfection cells and recombinant expression experiments.

According to the Lipofectamine 2000 kit instructions, 2.5 μg pcDNA-H5HA plasmid, 0.5 μg pcDNA-N1NA plasmid and 10 μl Lipofectamine 2000 transfection complex were transfected into a 35 mm diameter petri dish containing human cells with a growth density of about 80%-90% fusion rate. For embryonic kidney HEK-239 cells, the transfection complex was removed 6 hours after transfection, and fresh complete medium (DMEM medium containing 10% fetal bovine serum) was replaced.

After transfection, HEK-239 cells can transiently recombinantly express HA and NA, that is, cells that transiently recombinantly express HA and NA of influenza virus can be obtained.

For further optimization, cells with stable recombinant expression of HA and NA 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, supplemented with complete medium to 3ml, after overnight attachment, add G418 to a concentration of 600μg/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. The cells in the 96-well plate used for detection were fixed with 4% paraformaldehyde at room temperature for 5 minutes, washed with PBS, and Cy3 fluorescently labeled anti-H5 subtype HA antibody and FITC fluorescently labeled anti-N1 subtype NA antibody were added. For immunofluorescence detection, select wells with strong red and green fluorescent positive signals and a high proportion of red and green fluorescent positive cells, and continue to repeat the cloning and immunofluorescence of cells until the proportion of positive cells is close to 100%, and stable recombinant expression of the Cells of strain HA and NA.

Transient or stable recombinant expression of HA and NA cells of the strain, after adherent or suspension culture, the cells were blown and dispersed with PBS containing 2% BSA, the cell suspension was centrifuged, and the supernatant was removed and then washed with 0.1% BSA resuspend in PBS at a cell density of 10 ⁷ cells/ml. Add 2μl of 5mmol/L CFSE mother solution to each ml of cell suspension, place at 37°C for 10 minutes, then add 5ml of ice-cold PBS, place in the ice-water mixture for 5 minutes, centrifuge to remove the supernatant, and use an equal volume of Resuspend in PBS, wash by centrifugation twice, add an equal volume of PBS again, add an equal volume of 10% cold formaldehyde, and fix 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 105 cells/ml in PBS buffer containing 0.2% Nepalese gold ester, 2% BSA, 20% glycerol, and 0.5% heparin, so that it could be stored stably for a long time, and obtained Fluorescein CFSE-labeled recombinant eukaryotic cells expressing HA and NA antigens.

25 μl of the green fluorescent protein-labeled recombinant eukaryotic cell suspension expressing HA and NA antigens and 25 μl of the sample to be tested were mixed in a microagglutination plate, left at room temperature for 60 minutes, and then observed under a fluorescent microscope. If no agglutination occurs, the green fluorescent cells will settle at the bottom of the agglutination plate, and the fluorescent cells can be seen gathered in the center under the microscope; if agglutination occurs, the green fluorescent cells will be suspended in the liquid in flocculent form, and the fluorescent cells can be seen under the microscope Scattered across the field of view, clumping from cell to cell. Utilizing this feature, it is also possible to perform imaging with a fluorescence microscope, and use image analysis software to realize whether the software judges agglutination or not by setting up the fluorescent light-emitting area. When agglutination occurs in the sample to be tested, it indicates that the sample to be tested contains influenza virus HA and/or NA antibodies reactive with the strain HA and/or NA.

Example 3 Green Fluorescent Protein and Red Fluorescent Protein Labeling Method for Separately Recombined Eukaryotic Cells Expressing Influenza Virus HA and NA

In this example, an H5-type 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 sequences of its HA and NA can be obtained on the public database GenBank, and the sequence of HA The number is EU874899.2, and the serial number of NA is EU874900.2. 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.

According to the gene sequence of the strain NA, a pair of primers for NA full-length gene cloning was designed. 3 protective bases, Hind III restriction site, KOZAK sequence, start codon and pairing region; downstream primer N1NA-R, the sequence is: 5'-CTCGGATCCCTACTTGTCAATGGTGAATG-3', 3 in order from the 5' end Protective bases, BamH I restriction sites, stop codons and pairing regions. Utilize this pair of primers, carry out PCR amplification to virus cDNA with high-fidelity Taq enzyme, amplify NA fragment.

After the PCR product was recovered by agarose gel electrophoresis, after the HA gene fragment was recovered, it was digested with the red fluorescent expression vector using Hind III and BamH I respectively. to connect. The red fluorescent expression vector has a ribosome entry site sequence (IRES) between the encoded red fluorescent protein and the multiple cloning site. The HA gene fragment is inserted behind the promoter of the vector and before the IRES. When the promoter initiates transcription, it can Obtain an mRNA encoding HA, IRES and red fluorescent protein, and finally translate to obtain separate HA and red fluorescent protein. The NA fragment and the green fluorescent expression vector were digested with Hind III and BamH I, and recovered by electrophoresis for connection. There is a ribosome entry site sequence (IRES) between the green fluorescent protein encoded by the vector and the multiple cloning site, and the NA gene After the fragment is inserted into the promoter of the vector and before the IRES, an mRNA encoding NA, IRES and green fluorescent protein can be obtained when the promoter initiates transcription, and finally translated to obtain separate NA and green fluorescent protein.

The ligation products were respectively transformed into Escherichia coli DH5a competent cells, and coated with Amp resistance plates. After the colonies were subjected to PCR, the plasmids were extracted for enzyme digestion identification and DNA sequencing to obtain the eukaryotic expression plasmid containing the HA gene fragment, namely pIRES-Red-H5HA, and the eukaryotic expression plasmid containing the HA gene fragment, namely pIRES-EGFP-N1NA. Bacteria containing the pIRES-Red-H5HA plasmid and the pIRES-EGFP-N1NA plasmid were cultured overnight in LB containing 50 μg/ml ampicillin (Amp), respectively, and the pIRES-Red-H5HA plasmid and pIRES-Red-H5HA plasmid were extracted with a high-purity plasmid extraction kit. The pIRES-EGFP-N1NA plasmid is used for subsequent transfection of cells and recombinant expression experiments.

According to the instructions of the Lipofectamine 2000 kit, transfect 3μg pIRES-Red-H5HA plasmid and 10μl Lipofectamine 2000 transfection complex into a 35mm diameter culture dish containing human embryonic kidney HEK-239 cells with a growth density of about 80%-90% confluence 6h after transfection, the transfection complex was removed, and fresh complete medium (DMEM medium containing 10% fetal bovine serum) was replaced.

After transfection, HEK-239 cells can transiently recombinantly express HA, that is, cells that transiently recombinantly express influenza virus HA labeled with red fluorescent protein can be obtained.

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. After digesting the resistant cell colony 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 observe the cell growth under a fluorescent microscope Red fluorescence, repeated cloning of the cells until the ratio of red fluorescent positive cells is close to 100%, then cells marked with red fluorescent protein and stably recombinantly expressing the strain HA can be obtained.

According to the instructions of Lipofectamine 2000 kit, transfect 3μg pIRES-EGFP-N1NA plasmid and 10μl Lipofectamine 2000 transfection complex into a 35mm diameter culture dish containing human embryonic kidney HEK-239 cells with a growth density of about 80%-90% confluence 6h after transfection, the transfection complex was removed, and fresh complete medium (DMEM medium containing 10% fetal bovine serum) was replaced.

After transfection, HEK-239 cells can transiently recombinantly express NA, that is, cells that transiently recombinantly express influenza virus NA labeled with green fluorescent protein can be obtained.

For further optimization, cells with stable recombinant expression of NA can be screened: after 72 hours of transfection, the transfected cells were dispersed by pipetting, 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. After digesting the resistant cell colony 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, observe the green fluorescence under a fluorescent microscope, Select wells with strong fluorescent positive signal and high proportion of fluorescent positive cells, and continue to repeat the cloning and immunofluorescence of the cells until the proportion of positive cells is close to 100%, then you can obtain green fluorescent protein-labeled cells that stably recombinantly express the strain NA .

The transiently or stably recombinant cells expressing the strain HA labeled with red fluorescent protein were cultured, and the cells were gently digested with a low concentration of trypsin (0.05%) for about 5 minutes, and the medium containing fetal bovine serum was added to terminate the reaction. The suspension was centrifuged, the supernatant was removed, and then resuspended with PBS containing 2% BSA to obtain a trypsin-treated cell suspension. 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. 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 in PBS buffer containing 0.2% Nepalese gold ester, 2% BSA, 20% glycerol, and 0.5% heparin at a concentration of 10 ⁵ cells/ml, so that it could be stored stably for a long time, that is, The treated recombinant eukaryotic cells expressing HA antigen were obtained.

Green fluorescent protein-labeled cells transiently or stably recombinantly expressing the strain NA, after adherence or suspension culture, the cells were blown and dispersed with PBS containing 2% BSA, the cell suspension was centrifuged, and the supernatant was removed and then used with Resuspend in PBS with 2% BSA at a cell density of 10 ⁷ cells/ml. Remove the supernatant, resuspend with an equal volume of PBS, wash by centrifugation twice, add an equal volume of PBS again, add an equal volume of 10% cold formaldehyde, and fix at 4°C for 2 hours. Wash 3 times by centrifugation and resuspension in PBS. Finally, the cell pellet was resuspended in PBS buffer containing 0.2% Nepalese gold ester, 2% BSA, 20% glycerol, and 0.5% heparin at a concentration of 10 ⁵ cells/ml, so that it could be stored stably for a long time, that is, The treated recombinant eukaryotic cells expressing NA antigen were obtained.

The eukaryotic cells expressing HA and NA recombinantly labeled with the above-mentioned green fluorescent protein and red fluorescent protein respectively can be used for detection. At the same time, the two can also be mixed according to a certain ratio. The mixing ratio is that the content of eukaryotic cells expressing recombinant HA is 10 %-95%, preferably 70%-80%, the content of eukaryotic cells expressing HA recombinantly is 5%-90%, preferably 20%-30%, the cells after mixing have the same detection effect as those of co-recombinantly expressing HA and NA Eukaryotic cells are similar.

Mix 25 μl of eukaryotic cell suspension expressing HA or NA recombinantly expressed with green fluorescent protein and 25 μl of eukaryotic cell suspension individually recombinantly expressed with HA or NA labeled with red fluorescent protein, and 25 μl of the sample to be tested, mix in a micro-agglutination plate, room temperature After standing for 60 minutes, observe under a fluorescent microscope. If no agglutination occurs, the green fluorescent cells will settle at the bottom of the agglutination plate, and the fluorescent cells can be seen gathered in the center under the microscope; if agglutination occurs, the green fluorescent cells will be suspended in the liquid in flocculent form, and the fluorescent cells can be seen under the microscope Scattered across the field of view, clumping from cell to cell. Utilizing this feature, it is also possible to perform imaging with a fluorescence microscope, and use image analysis software to realize whether the software judges agglutination or not by setting up the fluorescent light-emitting area. When agglutination occurs in the sample to be tested, it indicates that the sample to be tested contains influenza virus HA and/or NA antibodies reactive with the strain HA and/or NA.

The eukaryotic cell suspension labeled with red fluorescent protein recombinantly expressed HA antigen (set as A), the eukaryotic cell suspension labeled with green fluorescent protein recombinantly expressed NA antigen (set as B), the recombinantly expressed HA labeled with green fluorescent protein and Add 25 μl of the eukaryotic cell suspension of NA antigen (or the mixed suspension of eukaryotic cells expressing HA and NA separately labeled with green fluorescent protein and red fluorescent protein, set as C) to a clean microagglutination plate, Then add 25 μl of the samples to be tested (serum, bronchial lavage fluid, plasma, interstitial fluid, etc.) to three different cell suspensions, mix them, leave them at room temperature for 60 minutes, and observe them under a fluorescent microscope. If no agglutination occurs, the green fluorescent cells will settle at the bottom of the agglutination plate, and the fluorescent cells can be seen gathered in the center under the microscope; if agglutination occurs, the green fluorescent cells will be suspended in the liquid in flocculent form, and the fluorescent cells can be seen under the microscope Scattered across the field of view, clumping from cell to cell. Utilizing this feature, it is also possible to perform imaging with a fluorescence microscope, and use image analysis software to realize whether the software judges agglutination or not by setting up the fluorescent light-emitting area. When agglutination occurs in the sample to be tested, it indicates that the sample to be tested contains influenza virus HA and/or NA antibodies reactive with the strain HA and/or NA.

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 with the above cell suspension, it means that the sample to be tested does not contain influenza virus HA reactive with the strain HA and NA. and NA antibodies or antibody concentrations below the detection levels achievable by this method. Set up positive serum and negative serum controls. If the control sample meets the expected results and the sample to be tested agglutinates with the above cell suspension, it means that the sample to be tested contains influenza virus HA and NA reactive with the strain HA and NA. NA antibody.

Set up positive serum and negative serum controls. If the control sample meets the expected results, and the sample to be tested agglutinates with both cell suspension A and cell suspension C, but does not agglutinate with cell suspension B, it means that the sample to be tested has agglutination. Contains influenza virus HA antibodies reactive with the strain HA, but does not contain influenza virus NA antibodies reactive with the strain NA or the antibody concentration is lower than the detection concentration achievable by the method.

Set up positive serum and negative serum controls. If the control sample meets the expected results, and the sample to be tested agglutinates with both cell suspension B and cell suspension C, but does not agglutinate with cell suspension A, it means that the sample to be tested has agglutination. Contains influenza virus NA antibodies reactive with the strain NA but does not contain influenza virus HA antibodies reactive with the strain HA or the antibody concentration is lower than the detection concentration achievable by the method.

When agglutination occurs in the sample to be tested, it indicates that the sample to be tested contains influenza virus HA and/or NA antibodies reactive with the strain HA and/or NA. 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 HA and/or NA antigen in Example 2 Reaction to obtain the highest dilution of the sample that can agglutinate, which 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 differences and relative ratios of influenza virus HA and/or NA antibodies in different samples to be tested are compared horizontally.

A fluorescent micro-cell agglutination method for detecting influenza virus antibody sequence listing

SEQUENCE LISTING

<110> Shantou University School of Medicine

<120> A fluorescent micro-cell agglutination method for detecting influenza virus antibody

<130>

<160>5

<170>PatentIn version 3.2

<210>1

<211>12

<212>DNA

<213> Artificial sequence

<400>1

agcaaaagca gg 12

<210>2

<211>31

<212>DNA

<213> Artificial sequence

<400>2

gcaaagctta ccatggagaa aatacttctt c 31

<210>3

<211>34

<212>DNA

<213> Artificial sequence

<400>3

ctcggatcct taaatgcaaa ttctgcattg taag 34

<210>4

<211>30

<212>DNA

<213> Artificial sequence

<400>4

gcaaagctta ccatgaatcc aaatcagaag 30

<210>5

<211>29

<212>DNA

<213> Artificial sequence

<400>5

ctcggatccc tacttgtcaa tggtgaatg 29

Claims (2)

1. A fluorescent micro-cell agglutination method for detecting influenza virus antibodies, characterized in that the method is to transfect eukaryotic cells after the influenza virus hemagglutinin gene and neuraminidase gene clone, so that the eukaryotic cell surface recombinant expression Influenza virus hemagglutinin and neuraminidase protein, the recombinant expression cells are fluorescently labeled, the recombinant expressed hemagglutinin and neuraminidase protein on the surface of the cell and the influenza virus hemagglutinin antibody and neuraminidase of the sample to be tested Acidase antibody binding, agglutination reaction occurs, fluorescence detection analysis, according to whether the fluorescent area increases to determine whether there is influenza virus antibody in the sample to be tested; the eukaryotic cells are human embryonic kidney HEK-239 cells. 2. the fluorescent micro-cell agglutination method of detecting influenza virus antibody according to claim 1, is characterized in that the eukaryotic cells expressing influenza virus hemagglutinin and neuraminidase recombinedly express influenza virus hemagglutination simultaneously Hemagglutinin and neuraminidase, or eukaryotic cells recombinantly expressing influenza virus hemagglutinin and eukaryotic cells recombinantly expressing neuraminidase, respectively. 3. the fluorescent microcell agglutination method of detecting influenza virus antibody according to claim 1, is characterized in that described recombinant expression is transient expression or stable expression. 4. the fluorescent microcell agglutination method of detecting influenza virus antibody according to claim 1, it is characterized in that the influenza virus hemagglutinin and the neuraminidase protein expressed recombinantly on the eukaryotic cell surface are complete full-length influenza virus Hemagglutinin and neuraminidase proteins, or antigenic epitopes of influenza virus hemagglutinin and neuraminidase. 5. the fluorescent microcell agglutination method of detecting influenza virus antibody according to claim 1, it is characterized in that the hemagglutinin expressed recombinantly on the surface of said eukaryotic cell is H1, H2, H3, H4, H5 of influenza A virus , H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 subtypes, hemagglutinin of influenza B virus or influenza C virus. 6. the fluorescent micro-cell agglutination method of detecting influenza virus antibody according to claim 1, it is characterized in that the neuraminidase protein expressed recombinantly on the surface of said eukaryotic cell is N1, N2, N3, N4 of influenza A virus , N5, N6, N7, N8, N9 subtypes, a neuraminidase of an influenza B virus or an influenza C virus. 7. the fluorescent microcell agglutination method of detecting influenza virus antibody according to claim 1, is characterized in that described influenza virus hemagglutinin antibody and neuraminidase antibody are one in IgG, IgM, IgA, IgE, IgD species or a mixture of several species. 8. The fluorescent micro-cell agglutination method for detecting influenza virus antibodies according to claim 1, characterized in that the fluorescent marker is fluorescein, fluorescent protein or other substances capable of emitting fluorescence. 9. The fluorescent micro-cell agglutination method for detecting influenza virus antibodies according to claim 1, characterized in that the equipment used for the fluorescence detection and analysis is a fluorescence microscope, a fluorescence photometer or a multifunctional microplate detector.