CN102023214A - Method for detecting influenza virus antibody through agglutination reaction based on eukaryotic cell for recombinant expression on influenza virus hemagglutinin and neuraminidase - Google Patents
Method for detecting influenza virus antibody through agglutination reaction based on eukaryotic cell for recombinant expression on influenza virus hemagglutinin and neuraminidase Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a method for detecting an influenza virus antibody through agglutination reaction based on an eukaryotic cell for recombinant expression on influenza virus hemagglutinin and neuraminidase. The method comprises the following steps of: taking the eukaryotic cell for the recombinant expression on the influenza virus hemagglutinin and the neuraminidase as an agglutination reaction matrix, and detecting the influenza virus hemagglutinin antibody and the neuraminidase antibody in a sample to be detected through the recombinant expression of the influenza virus hemagglutinin and the neuraminidase in the agglutination reaction way. In the invention, the influenza virus hemagglutinin and the neuraminidase are co-expressed on the surface of the eukaryotic cell, thereby effectively avoiding self-coagulation to affect the agglutination reaction due to the combination of the hemagglutinin and a cell receptor; at the same time, the invention can more widely and rapidly detect the influenza virus antibody in different serotypes.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of detection method of Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase.
Background technology
The human or animal of particular serotype influenza infection, infection back recovery, subclinical infection and vaccine inoculation, flu virus hemagglutinin antibody can appear in its body fluid (as serum, bronchial perfusate, blood plasma, tissue fluid etc.), by detecting the antibody of particular serotype influenza virus, can judge, diagnose or make a definite diagnosis this people or this animal and whether infect the influenza virus of this particular serotype such as (comprise early infection, subclinical infection or infect that the back is recovered) or inoculated influenza virus vaccine or estimated it to the resistibility of particular serotype influenza virus etc.
Detect the method that whether has the Antibody of Influenza of a certain particular serotype in the sample to be checked at present, mainly contain blood clotting and suppress experiment (HAI) and neutralization experiment, be about to the influenza virus elder generation and sample mix to be checked of a certain particular serotype, add then in red blood cell or the sensitive cells, judge whether sample to be checked can suppress this viral hemagglutination or cytopathic effect is judged, its shortcoming is to need to use to have infective virus, and there is hidden danger in security.In the experiment that relates to virus, according to regulation and the suggestion of country about rules (State Council's " pathogenic microorganism laboratory bio-safety management rules ", Ministry of Public Health's " pathogenic microorganism register that infect in the human world ", " the highly pathogenic pathogenic microorganism laboratory and the experimental activity bio-safety of human world infection are examined management method ") and WHO, the operation that comprises people H2N2 and part avian influenza virus requires to carry out in BSL-III (P3) laboratory, therefore, it is very high that the carrying out of said method requires laboratory condition, is not suitable for on-the-spot the detection and the scale detection.Document also has report, utilize the pseudovirion of expression of influenza virus HA to replace having infective influenza virus and carry out the HAI experiment, but technology is too complicated.
The shortcoming of said method also is to detect the antibody of influenza virus HA.The influenza virus surface mainly exists 2 kinds of glycoprotein furcellas that antigenicity is stronger, be hemagglutinin NA and neuraminidase NA, after influenza infection or subclinical infection or vaccine inoculation, human or animal's body can all produce antibody at HA and NA, just so, influenza A virus in the influenza virus is divided into 16 hypotypes such as H1-H16 according to the antigenicity of HA, the antigenicity of NA is divided into 9 hypotypes such as N1-N9, various combination mode according to HA and NA hypotype in influenza A virus has formed numerous hypotypes, as H5N1 (being the combination of H5 hypotype and N1 hypotype).Therefore, as detecting Antibody of Influenza, except that need detect HA serotype, also need NA serotype is detected.At present NA serotype detection of antibodies is then needed to detect by the neuraminidase enzyme method that suppresses alive separately.
Except that classical H AI and neutralization experiment, the report that also useful ELISA method and latex agglutination experimental technique detect.The advantage of ELISA and latex agglutination is to detect the antibody of HA and NA.Wherein, the ELISA method is to utilize recombinant expressed and influenza virus HA and/or NA purifying to detect corresponding antibody, this need obtain Recombinant Protein Expression and the purifying that keeps antigentic specificity, and because influenza virus has numerous hypotypes, even the serum cross reaction intensity of hypotype of the same race differs, HA and NA antigen to different serotypes all carry out Recombinant Protein Expression and purifying, its workload is big, technological requirement is high, batch between poor stability.The latex agglutination method is for existing other method (HAI, neutralization experiment and ELISA method), and the reaction time, short, suitable scene was detected, but still came with some shortcomings.The latex agglutination experimental technique is on inertia latex particle surface with the influenza virus HA of recombinant expressed and purifying and/or NA albumen coupling, utilize agglutinating reaction to detect the influenza virus corresponding antibody, this needs the influenza virus HA and the NA of recombinant expressed and purifying equally, and need acquisition to keep the Recombinant Protein Expression and the purifying of antigentic specificity, detect the influenza virus HA and the NA antibody of different serotypes as needs, just need carry out recombinant expressed and purifying to the HA and the NA of different strains, the same with the ELISA method, exist workload big, the technological requirement height, problem such as poor stability between batch.
Because whether detect Antibody of Influenza infects people or this animal and (comprises early infection, subclinical infection or infection recovery afterwards etc.) influenza virus of this particular serotype, or inoculated influenza virus vaccine, or estimate that it is significant to resistibility of particular serotype influenza virus etc., and present HAI, the neutralization experiment, detection means such as ELISA and latex agglutination, maybe need to use and have infective virus, or experiment condition and equipment that need be specific, or complex process, or detect shortcomings such as length consuming time, therefore, a kind of easy, fast, technology is simple and easy to control relatively, can detect influenza virus HA simultaneously and the NA antibody detection method is with a wide range of applications.
Summary of the invention
The complex process that the objective of the invention is to exist, detect problems such as length consuming time, poor stability, a kind of detection method based on the Antibody of Influenza of the eukaryotic agglutinating reaction of recombinant expressed influenza virus hemagglutinin and neuraminidase of quick, easy, good stability is provided according to the method that is used for detecting Antibody of Influenza in the prior art.
This method above-mentioned purpose is achieved by the following technical programs:
The inventive method is with influenza virus hemagglutinin gene and Neuraminidase Gene clone back transfecting eukaryotic cells, make eukaryotic recombinant expressed influenza virus hemagglutinin in surface and neuraminic acid zymoprotein, with flu virus hemagglutinin antibody and the neuraminidase antibodies in the sample to be checked, agglutinating reaction takes place, and detects Antibody of Influenza in the sample to be checked according to whether agglutinating reaction takes place
Particularly, the present invention is based on the Antibody of Influenza detection method of the eukaryotic agglutinating reaction of recombinant expressed influenza virus hemagglutinin (HA) and neuraminidase (NA), cell with recombinant expressed influenza virus HA and NA is a carrier, NA can destroy the acceptor of cell surface HA, prevent that nonspecific autoagglutination from appearring in cell, utilize the antigenicity of recombinant expressed HA of cell surface and NA simultaneously,, detect the antibody that whether has corresponding influenza virus in the sample to be checked by agglutinating reaction.
Influenza virus comprises influenza A virus, influenza B virus and influenza virus C, wherein the HA of influenza A virus can be divided into 16 hypotypes, NA can be divided into 9 hypotypes, simultaneously, because sudden change and the evolution of influenza virus HA and NA, even the HA of same hypotype and NA, the power of its cross reactivity differs.Therefore, when detecting a certain particular serotype Antibody of Influenza, use method of the present invention, the eukaryotic of the HA of recombinant expressed this particular serotype strain and sample to be checked are carried out the aggegation experiment get final product.Above-mentioned influenza virus HA, be meant H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, the H16 hypotype of influenza A virus, and the hemagglutinin of influenza B virus, influenza virus C, can use HA or its part of a certain particular serotype according to the needs of testing goal.Above-mentioned influenza virus NA is meant N1, N2, N3, N4, N5, N6, N7, N8, N9 hypotype and the influenza B virus of influenza A virus, the neuraminidase of influenza virus C.Above-mentioned recombinant expressed influenza virus HA can be arbitrary array mode of above-mentioned different HA and NA with NA.
As a kind of preferred version, described flu virus hemagglutinin antibody and neuraminic acid enzyme antibody are one or more the potpourri among IgG, IgM, IgA, IgE, the IgD.
Carrying out extensively in this way, the Antibody of Influenza of serotype detects, then can carry out antigenicity analysis according to the HA and the NA of the purpose serotype strain that is detected, select for use antigenicity is conservative or antigenic cross property the is strong HA and the part of NA albumen to carry out recombinant expressed eukaryotic structure, utilize agglutinating reaction that the extensive Antibody of Influenza of serotype is detected.Above-mentioned influenza virus HA and NA, can be a certain influenza virus strain HA and NA or wherein a part of, also can be splicing or the mixing of HA with NA or wherein a part of or the different HA and the NA of the susceptible poison strain of various flows, also can be that influenza virus HA and NA are suddenlyd change and modify, also can be the natural non-existent sequence of synthetic, have the antigenicity of influenza virus HA and NA but its core is a recombination expression product.
Above-mentioned recombinant expressed influenza virus HA and the eukaryotic of NA, being meant has the genetic fragment of influenza virus HA and NA to import eukaryotic coding, at HA and the NA of the recombinant expressed influenza virus in eukaryotic surface.Eukaryotic can be yeast, mammalian cell, insect cell, and other eukaryotic, and recombinant expressed influenza virus HA and NA are positioned at cell membrane or cell membrane surface.Wherein, eukaryotic preferred mammal cell and insect cell, its recombinant expressed influenza virus hemagglutinin can have more near virus infections posttranslational modification that the human or animal obtains, steric configuration and antigenicity, and the atopic of itself and influenza virus corresponding antibodies is better.Further, recombinant expressed influenza virus HA of preferred mammal cell and NA.Recombinant expressed can be transient expression, also can be stably express.It can be DNA that coding has the genetic fragment of influenza virus HA and NA, also can be RNA, it can be the simple fragment that contains promoter and coded sequence, it also can be plasmid, it also can be viral vectors, it is encoding influenza virus HA and NA only, also can be simultaneously or other albumen of independent hybrid coding or polypeptide or peptide section, its objective is that the genetic fragment with encoding influenza virus HA and NA obtains recombinant expressed in eukaryotic.Import eukaryotic mode and can use electric shock, liposome transfection, virus-mediated etc., it is recombinant expressed to obtain that purpose is that the genetic fragment with encoding influenza virus HA and NA enters eukaryotic.Above-mentioned recombinant expressed HA and the eukaryotic of NA, its core is to obtain to have at cell surface expression the eukaryotic of influenza virus HA and NA, its objective is the antigenicity of utilizing expressed HA and NA, by the aggegation experiment, detects Antibody of Influenza.In one embodiment of the invention, announced the eukaryotic method of recombinant expressed influenza virus HA and NA.
As a kind of preferred version, above-mentioned recombinant expressed influenza virus HA and the eukaryotic of NA, as the particulate antigen matrix that is used for agglutinating reaction, it can be the eukaryotic of natural recombinant expressed influenza virus hemagglutinin, or through pancreatin processing, mechanical dispersion, enzymolysis disperse, technology such as fixing agent is fixed, stabilizing agent is handled, antiseptic is handled simultaneously or the eukaryotic of the recombinant expressed influenza virus hemagglutinin of selectivity after handling, better to make " agglutinating reaction matrix " to preserve, prevent self-solidifying, to increase the sensitivity and the stability of agglutinating reaction detection.In one embodiment of the invention, announced the eukaryotic disposal route of recombinant expressed influenza virus hemagglutinin HA and NA, to improve sensitivity and the stability that agglutinating reaction detects.
As a kind of preferred version, in the detection method of the present invention, described eukaryotic is yeast, mammalian cell, insect cell or other eukaryotic, and recombinant expressed influenza virus hemagglutinin is positioned at cell membrane or cell membrane surface.
As a kind of preferred version, the influenza virus hemagglutinin of described eukaryotic surface expression and the ratio of neuraminidase are: 1: 19～19: 1; Effect is better when ratio is 1: 3～9: 1, best results when ratio is 4: 1.
Compared with prior art, the present invention has following beneficial effect:
The present invention is that the cell with recombinant expressed influenza virus HA and NA is a carrier, utilizes agglutinating reaction, detects the antibody that whether has corresponding influenza virus in the sample to be checked.Utilize HA and NA gene or its fragment of RT-PCR clone particular serotype influenza virus, make up carrier for expression of eukaryon, transfecting eukaryotic cells, cell surface can exist recombinant expressed HA and NA, after hydroformylation was fixing, this cell promptly became the sensitization particle that HA and NA antigen are contained in the surface, after sample mix to be checked, as containing the antibody of influenza virus in the sample to be checked, promptly agglutinating reaction can take place at several minutes.This method does not relate to and has infective virion; Compare ELISA, HAI and neutralization and test required a few hours to a couple of days, the reaction of the cell agglutination of recombinant expressed HA and NA only needs several minutes, and the reaction time is quick; Utilize universal primer and carrier to clone and transfection expression, can obtain the sensitized cell particle of different HA and NA antigen, can detect the different serotypes influenza virus more widely the HA and the NA gene of different serotypes.
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
The structure of the eukaryon expression plasmid of embodiment 1 influenza virus HA and NA
Present embodiment is an example with the strain A/Chicken/Guangdong/1/2005 (H5N1) of a strain H5 hypotype, carries out the preparation of recombinant expressed influenza virus hemagglutinin HA eukaryotic expression cell.A/Chicken/Guangdong/1/2005 (H5N1) is the strain H5N1 subtype influenza virus that separation obtains in the chicken in Guangdong Province in 2005, the gene order of its HA and NA can obtain on public database GenBank, the sequence number of HA is EU874899.2, and the sequence number of NA is EU874900.2.Use Viral RNA Miniprep Kit to extract viral RNA this strain virus propagation back, carry out reverse transcription, obtain viral cDNA with Uni-12 primer (5 '-AGCAAAAGCAGG-3 ') and SuperScript III or M-MLV reverse transcriptase.Gene order according to this strain HA, design a pair of primer that is used for HA full-length gene clone, its primer sequence is specific as follows: upstream primer H5HA-F, sequence is 5 '-GCAAAGCTTACCATGGAGAAAATAGTACTTCTTC-3 ', is followed successively by 3 protectiveness bases, Hind III restriction enzyme site, KOZAK sequence, initiation codon and collochore from 5 ' end; Downstream primer H5HA-R, sequence is 5 '-CTCGGATCCTTAAATGCAAATTCTGCATTGTAAG-3 ', is followed successively by 3 protectiveness bases, BamH I restriction enzyme site, terminator codon and collochore from 5 ' end.Utilize this to primer, viral cDNA is carried out pcr amplification, amplification HA fragment with high-fidelity Taq enzyme.Gene order according to this strain NA, design a pair of primer that is used for NA full-length gene clone, its primer sequence is specific as follows: upstream primer N1NA-F: sequence is 5 '-GCAAAGCTTACCATGAATCCAAATCAGAAG-3 ', is followed successively by 3 protectiveness bases, Hind III restriction enzyme site, KOZAK sequence, initiation codon and collochore from 5 ' end; Downstream primer N1NA-R, sequence is: 5 '-CTCGGATCCCTACTTGTCAATGGTGAATG-3 ' is followed successively by 3 protectiveness bases, BamH I restriction enzyme site, terminator codon and collochore from 5 ' end.Utilize this to primer, viral cDNA is carried out pcr amplification, amplification NA fragment with high-fidelity Taq enzyme.
The PCR product is after HA and NA genetic fragment after the agarose gel electrophoresis recovery, use Hind III and BamH I double digestion respectively with expression vector pcDNA3, utilize after electrophoresis reclaims once more that HA fragment after the T4DNA ligase cuts back to close enzyme is connected with carrier, the NA fragment is connected with carrier, transformed into escherichia coli DH5a competent cell is coated with the Amp resistant panel respectively.Bacterium colony extracts plasmid enzyme restriction and identifies and dna sequencing that the eukaryon expression plasmid that acquisition contains the HA genetic fragment is pcDNA-H5HA behind PCR, and the eukaryon expression plasmid that acquisition contains the HA genetic fragment is pcDNA-N1NA.The bacterium that contains pcDNA-H5HA plasmid and pcDNA-N1NA plasmid, respectively after the LB overnight incubation of the ampicillin through containing 50 μ g/ml (Amp), extract pcDNA-H5HA plasmid and pcDNA-N1NA plasmid with the high-purity plasmid extraction kit, be used for follow-up transfectional cell and recombinant expressed experiment.
The eukaryotic preparation of recombinant expressed influenza virus HA of 2 while of embodiment and NA
According to Lipofectamine 2000 kit explanations, with 2.5 μ g pcDNA-H5HA plasmids, 0.5 μ gpcDNA-N1NA plasmid and 10 μ l Lipofectamine, 2000 transfection composites, a 35mm diameter of transfection double dish contains Human Embryonic Kidney HEK-239 cell that stand density is about the 80%-90% fusion rate, 6h removes transfection composite after the transfection, changes fresh complete medium (the DMEM nutrient culture media that contains 10% hyclone).
After the transfection, the HEK-239 cell gets final product instantaneous recombinant expressed HA and NA, promptly obtains the cell of instantaneous recombinant expressed influenza virus HA and NA.
Be further optimization, can screen the cell that obtains to stablize recombinant expressed HA and NA: behind transfection 72h, transfectional cell piping and druming is disperseed, in new 35mm diameter double dish of 1/20 inoculation with cell suspension, replenish complete medium to 3ml, through spend the night adherent after, add G418 to concentration be 600 μ g/ml.After this per 3 to 5 days replacing once contains the complete medium of 600 μ g/ml G418, the drug-resistant colonies that about 3 visible screenings in week back occur.Drug-resistant colonies after trypsinization, is inoculated 96 orifice plates by 10-20 cell/ml and is cultivated, add the complete medium that contains 600 μ g/ml G418 be cultured to grow to individual layer after, go down to posterity, 96 orifice plates are cultivated in the backup that obtains to be used to detect.The cell that detects 96 orifice plates of usefulness is fixed 5 minutes with 4% paraformaldehyde room temperature, after the PBS rinsing, add the antibody of the fluorescently-labeled anti-H5 hypotype HA of Cy3 and the antibody of the fluorescently-labeled anti-N1 hypotype NA of FITC and carry out the immunofluorescence detection, select red and the green fluorescence positive signal by force, redness and the high hole of gfp positive cell ratio, pair cell continues the cloning and the immunofluorescence of repetition, to the positive cell ratio near 100%, can obtain stablizing the cell of recombinant expressed this strain HA and NA.
The eukaryotic processing of recombinant expressed influenza virus HA of 3 while of embodiment and NA
Instantaneous or stablize the cell of recombinant expressed this strain HA and NA, after adherent or suspension cultured, cell piping and druming is disperseed with the PBS that contains 2%BSA, cell suspension is carried out centrifugal, remove behind the supernatant with containing the PBS of 2%BSA by 10
7Individual/ml cell density carries out resuspended.Remove supernatant, wash 2 times, add isopyknic PBS once more, add isopyknic 10% cold formaldehyde, fix 2 hours in 4 ℃ with isopyknic PBS is resuspended, centrifugal.Carry out 3 washings through the mode centrifugal, that PBS is resuspended.At last with cell precipitation, by 10
7The concentration of individual/ml is resuspended in the PBS damping fluid that contains 0.2% Nepal gold ester, 2%BSA, 20% glycerine, 0.5% heparin, and it can stably be preserved for a long time, the recombinant expressed HA after promptly having obtained handling and the eukaryotic of NA antigen.
Embodiment 4 uses the eukaryotic of common recombinant expressed influenza virus HA and NA to carry out agglutinating reaction and detects Antibody of Influenza
The eukaryotic suspension 1 (about 50 μ l) of recombinant expressed HA after the processing among the embodiment 2 and NA antigen is added on the clean microslide, the sample to be checked (serum, bronchial perfusate, blood plasma, tissue fluid etc.) that adds 50 μ l then, rock microslide all around gently or use micropipette tip, observed cell agglutination whether occurs in 1-3 minute under the room temperature its mixing.
Set up the contrast of positive serum and negative serum, meet expected results as control sample, when not aggegation takes place sample to be checked, illustrate not contain in the sample to be checked to have reactive influenza virus HA and/or NA antibody or antibody concentration with this strain HA and/or NA and be lower than the detectable concentration that this method can reach.
During sample generation aggegation to be checked, illustrate to contain in the sample to be checked to have reactive influenza virus HA and/or NA antibody with this strain HA and/or NA.Can further carry out relative quantitative assay this moment, promptly, sample 1 to be checked is carried out continuous doubling dilution, then respectively with embodiment 2 in processing after recombinant expressed HA and/or the eukaryotic suspension of NA antigen carry out agglutinating reaction, obtain to take place the high dilution of sample of aggegation, be made as d1.Can obtain the high dilution d2 of the agglutinating reaction positive of sample 2 to be checked with quadrat method.Concentration difference and relative scale with influenza virus HA and/or NA antibody in the different samples to be checked of this lateral comparison.
The eukaryotic preparation of embodiment 5 independent recombinant expressed influenza virus HA and NA
According to Lipofectamine 2000 kit explanations, with 3 μ g pcDNA-H5HA plasmids and 10 μ lLipofectamine, 2000 transfection composites, a 35mm diameter of transfection double dish contains Human Embryonic Kidney HEK-239 cell that stand density is about the 80%-90% fusion rate, 6h removes transfection composite after the transfection, changes fresh complete medium (the DMEM nutrient culture media that contains 10% hyclone).
After the transfection, the HEK-239 cell gets final product instantaneous recombinant expressed HA, promptly obtains the cell of instantaneous recombinant expressed influenza virus HA.
Be further optimization, can screen the cell that obtains to stablize recombinant expressed HA: behind transfection 72h, transfectional cell piping and druming is disperseed, in new 35mm diameter double dish of 1/20 inoculation with cell suspension, replenish complete medium to 3ml, through spend the night adherent after, add G418 to concentration be 600 μ g/ml.After this per 3 to 5 days replacing once contains the complete medium of 600 μ g/ml G418, the drug-resistant colonies that about 3 visible screenings in week back occur.Drug-resistant colonies after trypsinization, is inoculated 96 orifice plates by 10-20 cell/ml and is cultivated, add the complete medium that contains 600 μ g/ml G418 be cultured to grow to individual layer after, go down to posterity, 96 orifice plates are cultivated in the backup that obtains to be used to detect.The cell that detects 96 orifice plates of usefulness is fixed 5 minutes with 4% paraformaldehyde room temperature, after the PBS rinsing, the antibody that adds anti-H5 hypotype HA is that one anti-, Cy3 fluorescently-labeled two anti-immunofluorescences of carrying out detect, select the hole that the fluorescence positive signal is strong, the fluorescencepositive cell ratio is high, pair cell continues the cloning and the immunofluorescence of repetition, to the positive cell ratio near 100%, can obtain stablizing the cell of recombinant expressed this strain HA.
According to Lipofectamine 2000 kit explanations, with 3 μ g pcDNA-N1NA plasmids and 10 μ lLipofectamine, 2000 transfection composites, a 35mm diameter of transfection double dish contains Human Embryonic Kidney HEK-239 cell that stand density is about the 80%-90% fusion rate, 6h removes transfection composite after the transfection, changes fresh complete medium (the DMEM nutrient culture media that contains 10% hyclone).
After the transfection, the HEK-239 cell gets final product instantaneous recombinant expressed NA, promptly obtains the cell of instantaneous recombinant expressed influenza virus NA.
Be further optimization, can screen the cell that obtains to stablize recombinant expressed NA: behind transfection 72h, transfectional cell piping and druming is disperseed, in new 35mm diameter double dish of 1/20 inoculation with cell suspension, replenish complete medium to 3ml, through spend the night adherent after, add G418 to concentration be 600 μ g/ml.After this per 3 to 5 days replacing once contains the complete medium of 600 μ g/ml G418, the drug-resistant colonies that about 3 visible screenings in week back occur.Drug-resistant colonies after trypsinization, is inoculated 96 orifice plates by 10-20 cell/ml and is cultivated, add the complete medium that contains 600 μ g/ml G418 be cultured to grow to individual layer after, go down to posterity, 96 orifice plates are cultivated in the backup that obtains to be used to detect.The cell that detects 96 orifice plates of usefulness is fixed 5 minutes with 4% paraformaldehyde room temperature, after the PBS rinsing, the antibody that adds anti-N1 hypotype NA is that one anti-, Cy3 fluorescently-labeled two anti-immunofluorescences of carrying out detect, select the hole that the fluorescence positive signal is strong, the fluorescencepositive cell ratio is high, pair cell continues the cloning and the immunofluorescence of repetition, to the positive cell ratio near 100%, can obtain stablizing the cell of recombinant expressed this strain NA.
The eukaryotic processing of embodiment 6 independent recombinant expressed influenza virus HA and NA
Instantaneous or stablize the cell of recombinant expressed this strain HA, after cultivating, with about 5 minutes of the gentle vitellophag of low concentration pancreatin (0.05%), add the nutrient culture media cessation reaction that contains hyclone, carry out cell suspension centrifugal, it is resuspended to use the PBS that contains 2%BSA to carry out behind the removal supernatant, obtains the cell suspension after pancreatin is handled.For preventing that cell that HA may cause is from aggegation, add neuraminidase in the cell suspension after pancreatin is handled and (claim neuraminidase again, Neuraminidase) handle, eliminate the effect of recombinant expressed HA and cell autoreceptor, avoid cell autoagglutination.Promptly, with cell suspension after centrifugal, with 50mM sodium citrate (pH 6.0) damping fluid, undertaken resuspended by 107/ml cell, the neuraminidase that adds 200 units in every ml cell suspension, centrifugal after handling 30 minutes under 37 ℃ of conditions, remove supernatant, wash 2 times with isopyknic PBS is resuspended, centrifugal, add isopyknic PBS once more.The above-mentioned cell suspension of handling through pancreatin and neuraminidase adds isopyknic 10% cold formaldehyde, fixes 2 hours in 4 ℃.Carry out 3 washings through the mode centrifugal, that PBS is resuspended.At last with cell precipitation, by 10
7The concentration of individual/ml is resuspended in the PBS damping fluid that contains 0.2% Nepal gold ester, 2%BSA, 20% glycerine, 0.5% heparin, and it can stably be preserved for a long time, the eukaryotic of the recombinant expressed HA antigen after promptly having obtained handling.Instantaneous or stablize the cell of recombinant expressed this strain NA, after adherent or suspension cultured, cell piping and druming is disperseed with the PBS that contains 2%BSA, cell suspension is carried out centrifugal, remove behind the supernatant with containing the PBS of 2%BSA by 10
7Individual/ml cell density carries out resuspended.Remove supernatant, wash 2 times, add isopyknic PBS once more, add isopyknic 10% cold formaldehyde, fix 2 hours in 4 ℃ with isopyknic PBS is resuspended, centrifugal.Carry out 3 washings through the mode centrifugal, that PBS is resuspended.At last with cell precipitation, by 10
7The concentration of individual/ml is resuspended in the PBS damping fluid that contains 0.2% Nepal gold ester, 2%BSA, 20% glycerine, 0.5% heparin, and it can stably be preserved for a long time, the eukaryotic of the recombinant expressed NA antigen after promptly having obtained handling.
The eukaryotic of above-mentioned independent recombinant expressed HA and NA is available detection, also both can be mixed by a certain percentage simultaneously, blending ratio is that the eukaryotic content of recombinant expressed HA is 10%-95%, preferred 70%-80%, the eukaryotic content of recombinant expressed HA is 5%-90%, preferred 20%-30%, mixed cell is similar with the eukaryotic of common recombinant expressed HA and NA on the detection effect.
That embodiment 6 uses is common, separately and mix the agglutinating reaction that the eukaryotic of recombinant expressed influenza virus HA and NA is optimized and detect Antibody of Influenza
Eukaryotic suspension (being made as A) with recombinant expressed HA antigen, the eukaryotic suspension (being made as B) of recombinant expressed NA antigen, the eukaryotic suspension of recombinant expressed HA and NA antigen (or the eukaryotic mixing suspension of single expression HA and NA, be made as C), respectively adding 1 (about 50 μ l) is added on the clean microslide, sample (the serum to be checked that respectively adds 50 μ l then, bronchial perfusate, blood plasma, tissue fluid etc.) to 3 kinds of different cell suspensions, rock microslide all around gently or use micropipette tip, observed cell agglutination whether occurs in 1-3 minute under the room temperature its mixing.
Set up the contrast of positive serum and negative serum, meet expected results as control sample, sample to be checked takes place and above-mentioned cell suspension all not during aggegation, illustrates not contain in the sample to be checked to have reactive influenza virus HA and NA antibody or antibody concentration with this strain HA and NA and be lower than the detectable concentration that this method can reach.Set up the contrast of positive serum and negative serum, meet expected results, when sample generation to be checked all aggegation takes place with above-mentioned cell suspension, illustrate to contain in the sample to be checked to have reactive influenza virus HA and NA antibody with this strain HA and NA as control sample.
Set up the contrast of positive serum and negative serum, meet expected results as control sample, when aggegation all takes place with cell suspension A and cell suspension C in sample generation to be checked, and when with cell suspension B aggegation taking place, illustrate to contain in the sample to be checked to have reactive influenza virus HA antibody, do not have reactive influenza virus NA antibody with this strain NA or antibody concentration is lower than the detectable concentration that this method can reach and do not contain with this strain HA.
Set up the contrast of positive serum and negative serum, meet expected results as control sample, when aggegation all takes place with cell suspension B and cell suspension C in sample generation to be checked, and when with cell suspension A aggegation taking place, illustrate to contain in the sample to be checked to have reactive influenza virus NA antibody, do not have reactive influenza virus HA antibody with this strain HA or antibody concentration is lower than the detectable concentration that this method can reach and do not contain with this strain NA.
During sample generation aggegation to be checked, illustrate to contain in the sample to be checked to have reactive influenza virus HA and/or NA antibody with this strain HA and/or NA.Can further carry out relative quantitative assay this moment, promptly, sample 1 to be checked is carried out continuous doubling dilution, then respectively with embodiment 2 in processing after recombinant expressed HA and/or the eukaryotic suspension of NA antigen carry out agglutinating reaction, obtain to take place the high dilution of sample of aggegation, be made as d1.Can obtain the high dilution d2 of the agglutinating reaction positive of sample 2 to be checked with quadrat method.Concentration difference and relative scale with influenza virus HA and/or NA antibody in the different samples to be checked of this lateral comparison.
Antibody of Influenza detection method sequence table based on the eukaryotic agglutinating reaction of recombinant expressed influenza virus hemagglutinin and neuraminidase
SEQUENCE?LISTING
＜110〉Medical College of Shantou University
＜120〉based on the influenza virus of the eukaryotic agglutinating reaction of recombinant expressed influenza virus hemagglutinin and neuraminidase
Antibody detection method
<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>34
<212>DNA
＜213〉artificial sequence
<400>2
gcaaagctta?ccatggagaa?aatagtactt?cttc 34
<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 (10)
1. detection method based on the Antibody of Influenza of the eukaryotic agglutinating reaction of recombinant expressed influenza virus hemagglutinin and neuraminidase, it is characterized in that described method is with influenza virus hemagglutinin gene and Neuraminidase Gene clone back transfecting eukaryotic cells, make eukaryotic recombinant expressed influenza virus hemagglutinin in surface and neuraminic acid zymoprotein, flu virus hemagglutinin antibody and neuraminidase antibodies in agglutinating reaction matrix and the sample to be checked, agglutinating reaction takes place, and detects Antibody of Influenza in the sample to be checked according to whether agglutinating reaction takes place.
2. the detection method of the Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase according to claim 1, it is characterized in that described agglutinating reaction matrix is the natural recombinant expressed influenza virus hemagglutinin and the eukaryotic of neuraminidase, or through pancreatin processing, mechanical dispersion, enzymolysis disperses, fixing agent is fixed, stabilizing agent is handled, antiseptic is handled method simultaneously or the recombinant expressed influenza virus hemagglutinin after the selectivity processing and the eukaryotic of neuraminidase.
3. the detection method of the Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase according to claim 1, the eukaryotic that it is characterized in that described recombinant expressed influenza virus hemagglutinin and neuraminidase is common recombinant expressed influenza virus hemagglutinin of while and neuraminidase, and the eukaryotic of perhaps distinguishing recombinant expressed influenza virus hemagglutinin mixes with the eukaryotic of recombinant expressed neuraminidase.
4. the detection method of the Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase according to claim 1, it is characterized in that influenza virus hemagglutinin and neuraminic acid zymoprotein that influenza virus hemagglutinin that described eukaryotic surface is recombinant expressed and neuraminic acid zymoprotein are complete total length, or the part of influenza virus hemagglutinin albumen and neuraminidase, or the epitope of influenza virus hemagglutinin and neuraminidase, or the partial amino-acid in influenza virus hemagglutinin and neuraminic acid zymoprotein total length or the partial peptide section suddenlyd change or modify, or influenza virus hemagglutinin and neuraminic acid zymoprotein and other albumen, polypeptide, the peptide section is carried out amalgamation and expression or common independent the expression.
5. the detection method of the Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase according to claim 1 is characterized in that described recombinant expressed be transient expression or stably express.
6. the detection method of the Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase according to claim 1 is characterized in that the recombinant expressed hemagglutinin in described eukaryotic surface is the hemagglutinin of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 hypotype, influenza B virus or the influenza virus C of influenza A virus.
7. the detection method of the Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase according to claim 1 is characterized in that the recombinant expressed neuraminic acid zymoprotein in described eukaryotic surface is the neuraminidase of N1, N2, N3, N4, N5, N6, N7, N8, N9 hypotype, influenza B virus or the influenza virus C of influenza A virus.
8. the detection method of the Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase according to claim 1 is characterized in that described flu virus hemagglutinin antibody and neuraminic acid enzyme antibody are one or more the potpourri among IgG, IgM, IgA, IgE, the IgD.
9. the detection method of the Antibody of Influenza of the eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase according to claim 1, it is characterized in that described eukaryotic is yeast, mammalian cell, insect cell or other eukaryotic, recombinant expressed influenza virus hemagglutinin is positioned at cell membrane or cell membrane surface.
10. according to the detection method of the Antibody of Influenza of the described eukaryotic agglutinating reaction based on recombinant expressed influenza virus hemagglutinin and neuraminidase of any claim in the claim 1～9, it is characterized in that the influenza virus hemagglutinin of described eukaryotic surface expression and the ratio of neuraminidase are 1: 19～19: 1.
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| CN105486871A (en) * | 2015-11-25 | 2016-04-13 | 北京世纪元亨动物防疫技术有限公司 | Colloidal gold test strip for rapidly detecting hemagglutination inhibition titer of canine parvovirus antibody, and kit and detection method thereof |
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| CN101448934A (en) * | 2006-03-23 | 2009-06-03 | 日本赤十字社 | Panel cell used for granulocyte antibody detection |
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| CN105486871A (en) * | 2015-11-25 | 2016-04-13 | 北京世纪元亨动物防疫技术有限公司 | Colloidal gold test strip for rapidly detecting hemagglutination inhibition titer of canine parvovirus antibody, and kit and detection method thereof |
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