CN102023213B - Fluorescence micro cell agglutination method for detecting influenza virus antibody - Google Patents
Fluorescence micro cell agglutination method for detecting influenza virus antibody Download PDFInfo
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Abstract
The invention relates to the biological technology field, in particular to a fluorescence micro cell agglutination method for detecting influenza virus antibody. The method in the invention comprises the following steps: hemagglutinin gene and neuraminidase gene of influenza virus are cloned and then are transfected to a eukaryotic cell; the hemagglutinin and neuraminidase are recombined and expressed on the surface of the eukaryotic cell; the recombinant expression cell is marked by fluorescence; the hemagglutinin and neuraminidase which are recombined and expressed on the surface of the cell are combined with hemagglutinin antibody and neuraminidase antibody of the influenza virus to carry out agglutination reaction; and pictures are taken by a fluorescence microscope, whether influenza virus antibody exists in a to-be-detected sample is judged according to whether the fluorescence area is increased. In the detecting method of the invention, the cell marked by fluorescence is used, the cell using amount is reduced, high-throughput detection can be carried out, and quick detection, early detection, on-spot detection and evaluation of the influenza virus antibody can be realized.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of fluorescence few cells aggegation method that detects Antibody of Influenza.
Background technology
The human or animal of recovery after particular serotype influenza infection, infection, subclinical infection and vaccine inoculation, in its body fluid (as serum, bronchial perfusate, blood plasma, tissue fluid etc.), there will be flu virus hemagglutinin antibody, 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 (comprise early infection, subclinical infection or infect then recover etc.) this particular serotype or inoculated influenza virus vaccine or evaluated it to the resistibility of particular serotype influenza virus etc.
Detect at present the method that whether has the Antibody of Influenza of a certain particular serotype in sample to be checked, 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, then add in red blood cell or 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 security exists hidden danger.In relating to viral experiment, according to national regulations (State Council's < < pathogenic microorganism laboratory Biosafety management regulations > >, the pathogenic microorganism register > > that infect in the < < of the Ministry of Public Health human world, highly pathogenic pathogenic microorganism laboratory and the experimental activity bio-safety that infect in the < < human world are examined management method > >) and regulation and the suggestion of WHO, the operation requirements that comprises people H2N2 and part avian influenza virus carries out in BSL-III (P3) laboratory, therefore, it is very high that the carrying out of said method requires laboratory condition, not being suitable for Site Detection and scale detects.Document also has report, utilize the pseudovirion of expression of influenza virus HA to replace having infective influenza virus and carry out HAI experiment, but technology is too complicated.
The shortcoming of said method is also to detect the antibody of influenza virus HA.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 for HA and NA, just so, influenza A virus in 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, in influenza A virus, according to the various combination mode of HA and NA hypotype, numerous hypotypes have been formed, as H5N1 (being H5 hypotype and the combination of N1 hypotype).Therefore,, as detected Antibody of Influenza, except need detect HA serotype, also need NA serotype to detect.At present the detection of NA serotype antibody is needed by the neuraminidase enzyme method suppressing alive, to detect separately.
Except classical HAI 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, ELISA method is to utilize recombinant expressed and influenza virus HA and/or NA purifying to detect corresponding antibody, this need to obtain the expression and purification of the recombinant protein that keeps antigentic specificity, and because influenza virus has numerous hypotypes, even if the serum cross reaction intensity of hypotype of the same race differs, the HA of different serotypes and NA antigen are carried out to the expression and purification of recombinant protein, its workload is large, technological requirement is high, batch between poor stability.Latex agglutination method is for existing other method (HAI, neutralization experiment and ELISA method), and the reaction time is short, be applicable to Site Detection, but still comes with some shortcomings.Latex agglutination experimental technique is that the influenza virus HA of recombinant expressed and purifying and/or NA albumen coupling is surperficial at inertia latex particle, utilize agglutinating reaction to detect the corresponding antibody of influenza virus, this needs influenza virus HA and the NA of recombinant expressed and purifying equally, and need acquisition to keep the expression and purification of the recombinant protein of antigentic specificity, as detected influenza virus HA and the NA antibody of different serotypes, just need to carry out recombinant expressed and purifying to the HA of different strains and NA, the same with ELISA method, exist workload large, technological requirement is high, the problem such as poor stability between batch.
Owing to detecting Antibody of Influenza, whether people or this animal are infected and (comprise early infection, subclinical infection or infect after recover etc.) influenza virus of this particular serotype, or inoculated influenza virus vaccine, or evaluate that it is significant to resistibility of particular serotype influenza virus etc., and current HAI, neutralization experiment, the detection means such as ELISA and latex agglutination, maybe need to use and there is infective virus, or the specific experiment condition of needs and equipment, or complex process, or detect the shortcomings such as length consuming time, therefore, a kind of easy, fast, technique is relatively simple and easy to control, influenza virus HA can be detected simultaneously and NA antibody detection method is with a wide range of applications.
Using the eukaryotic of recombinant expressed influenza virus hemagglutinin (HA) and neuraminidase (NA) is agglutinating reaction matrix, utilize agglutinating reaction, detect the antibody that whether has respective streams Influenza Virus in sample to be checked, this method, for above-mentioned classic method, can saferly detect Antibody of Influenza faster.But because the method exists the shortcoming that flux is limited, cell consumption amount is large, therefore, a kind of applicable high flux detection of needs, cell consumption are still less, easy, quick, technique is relatively simple and easy to control, can detect influenza virus HA and NA antibody detection method simultaneously.
Summary of the invention
The object of the invention is to according to shortcomings such as large for detection of the cell consumption amount existing in Antibody of Influenza in prior art, complex process, detection length consuming time, flux are limited, provide a kind of easy, quick, cell consumption is few, be applicable to the method for detection of Antibody of Influenza that high flux detects.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
The present invention is by transfecting eukaryotic cells after influenza virus hemagglutinin gene and Neuraminidase Gene clone, make the eukaryotic recombinant expressed influenza virus hemagglutinin in surface and neuraminic acid zymoprotein, restructuring express cell is carried out to fluorescence labeling, the recombinant expressed hemagglutinin of this cell surface and neuraminic acid zymoprotein are combined with flu virus hemagglutinin antibody and the neuraminic acid enzyme antibody of testing sample, there is agglutinating reaction, fluoroscopic examination is analyzed, and according to fluorescence area, whether increases to judge in testing sample, whether have Antibody of Influenza.
As a kind of preferred version, described fluorescence labeling is that fluorescein, fluorescin or other can send the material of fluorescence; It is fluorescent microscope, fluorophotometer or multi-functional microwell plate detector that equipment used is analyzed in described fluoroscopic examination.
Utilize HA and NA gene or its fragment of RT-PCR clone particular serotype influenza virus, build carrier for expression of eukaryon, transfecting eukaryotic cells, can there is recombinant expressed HA and NA in cell surface, after hydroformylation is fixing, this cell becomes the sensitization particle that HA and NA antigen are contained in surface.This cell nucleic acid of transfection coding fluorescence albumen (albumen or polypeptide or mutant that green fluorescent protein, enhanced green fluorescence protein, red fluorescent protein, yellow fluorescence protein, cyan fluorescent protein etc. can emitting fluorescences under exciting light) simultaneously when transfection, this cell can be by fluorescence labeling.Fluorescence labeling also can be used fluorescent material, as CFSE, SYTO, Cy3, Cy5, FITC etc., cell is carried out to mark.After cell after fluorescence labeling mixes in micro-agglutination plate with sample to be checked, as the antibody that contains influenza virus in sample to be checked, can there is agglutinating reaction at several minutes, can clear observation under fluorescent microscope and judge whether occur aggegation.Simultaneously also can be by image analysis software after the imaging of taking pictures, as Image Pro Plus etc. analyzes each hole fluorescence area, utilize cell to occur being difficult for after aggegation precipitation thereby fluorescence immunoassay compared with large and judge whether to occur aggegation.
Particularly, the technical solution used in the present invention is as follows:
By the eukaryotic of recombinant expressed influenza virus hemagglutinin (HA), or the eukaryotic of recombinant expressed neuraminidase (NA), or expression simultaneously has the eukaryotic of HA and NA, or the eukaryotic potpourri of independent recombinant expressed HA and NA, with fluorescent material, carry out mark, after cell after fluorescence labeling mixes in micro-agglutination plate with sample to be checked, as the antibody that contains influenza virus in sample to be checked, can there is agglutinating reaction at several minutes, can clear observation under fluorescent microscope and judge whether occur aggegation.Simultaneously also can be by image analysis software after the imaging of taking pictures, as Image Pro Plus etc. analyzes each hole fluorescence area, utilize cell to occur being difficult for after aggegation precipitation thereby fluorescence immunoassay compared with large and judge whether to occur aggegation.As there is aggegation, show to detect the antibody that whether has respective streams Influenza Virus in sample to be checked.
As a kind of preferred version, described recombinant expressed be transient expression or stably express.
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, sudden change and evolution due to influenza virus HA and NA, even the HA of same hypotype and NA, the power of its cross reactivity differs.Therefore, while 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 to aggegation experiment.Above-mentioned influenza virus HA, refer to 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 be according to the HA that need to use a certain particular serotype of testing goal or its part.Above-mentioned influenza virus NA, refers to 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 and NA can be arbitrary array modes of above-mentioned different HA and NA.
As a kind of preferred version, described flu virus hemagglutinin antibody and neuraminic acid enzyme antibody are one or more the potpourri in IgG, IgM, IgA, IgE, IgD.
The Antibody of Influenza of carrying out in this way more extensive serotype detects, can carry out antigenicity analysis according to HA and the NA of detected object serotype strain, select antigenicity is conservative or antigenic cross property is strong HA and a part for NA albumen to carry out recombinant expressed eukaryotic structure, utilize agglutinating reaction to detect the Antibody of Influenza of more extensive serotype.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 the HA of various flows Influenza Virus strain and NA or wherein splicing or the mixing of a part or different HA and NA, also can be that infected by influenza HA and NA suddenly change and modify, also can be artificial synthetic natural non-existent sequence, but its core is recombination expression product, there is the antigenicity of influenza virus HA and NA.
Above-mentioned recombinant expressed influenza virus HA and the eukaryotic of NA, refer to and have 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 and more approaches virus infections posttranslational modification that human or animal obtains, steric configuration and antigenicity, and the atopic of itself and influenza virus corresponding antibodies is better.Further, the recombinant expressed influenza virus HA of preferred mammal cell and NA.Recombinant expressed can be transient expression, can be also 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, also can be plasmid, 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, and its objective is the genetic fragment of encoding influenza virus HA and NA is obtained recombinant expressed in eukaryotic.Import eukaryotic mode and can use electric shock, liposome transfection, virus-mediated etc., object is that the genetic fragment of encoding influenza virus HA and NA is entered to eukaryotic is recombinant expressed to obtain.Above-mentioned recombinant expressed HA and the eukaryotic of NA, its core is to obtain the eukaryotic that has influenza virus HA and NA at cell surface expression, and this cell has been carried out fluorescence labeling by fluorescent material, its objective is and utilize fluorescence, can high flux ground with less cell concentration, can test detection Antibody of Influenza by aggegation.In embodiments of the invention, announced the eukaryotic method of fluorescently-labeled recombinant expressed influenza virus HA and NA.
Detection method of the present invention eukaryotic used is yeast, mammalian cell, insect cell or other eukaryotic, and recombinant expressed influenza virus hemagglutinin is positioned at cell membrane or cell membrane surface.Using eukaryotic to carry out recombinant expressed object is influenza virus hemagglutinin and neuraminic acid zymoprotein or its partial peptide section with posttranslational modification (as glycosylation etc.) of living, with better and one of flu virus hemagglutinin antibody and neuraminic acid enzyme antibody or both identify, combination and aggegation, obtain better compatibility and specificity.
Compared with prior art, the present invention has following beneficial effect:
The fluorescence few cells aggegation method that the present invention detects Antibody of Influenza 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 cell agglutination reaction of fluorescently-labeled recombinant expressed HA and NA is less than 60 minutes, and the reaction time is quick; Utilize universal primer and carrier to clone and transfection expression the HA of different serotypes and NA gene, can obtain the sensitized cell particle of different HA and NA antigen, can to different serotypes Antibody of Influenza, detect more widely; With respect to the recombinant expressed HA of non-fluorescent label and the cell agglutination reaction method of NA, the present invention is applicable to that high flux detects, cell consumption still less (only need do not use fluorescence labeled cell 1/100 to 1/10).
Embodiment
Below in conjunction with embodiment, further explain the present invention, but embodiment does not limit in any form to the present invention.
The recombinant expressed influenza virus HA of embodiment 1 Green Fluorescent Protein and the eukaryotic method of NA
It is example that the present embodiment be take 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 separated strain H5N1 subtype influenza virus obtaining in the chicken in 2005 Nian Guangdong Province, 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.To after this strain virus propagation, use Viral RNA Miniprep Kit to extract viral RNA, with Uni-12 primer (5 '-AgCAAAAgCAgg-3 ') and SuperScript III or M-MLV reverse transcriptase, carry out reverse transcription, obtain viral cDNA.According to the gene order of this strain HA, design a pair of primer for HA full-length gene clone, its primer sequence is specific as follows: upstream primer H5HA-F, sequence is 5 '-GCAAAGCTTACCATGGAGAAAATACTTCTTC-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, Hind III restriction enzyme site, KOZAK sequence, initiation codon and collochore; Downstream primer H5HA-R, sequence is 5 '-CTCGGATCCTTAAATGCAAATTCTGCATTGTAAG-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, BamH I restriction enzyme site, terminator codon and collochore.Utilize this to primer, with high-fidelity Taq enzyme, viral cDNA is carried out to pcr amplification, amplification HA fragment.
According to the gene order of this strain NA, design a pair of primer for NA full-length gene clone, its primer sequence is specific as follows: upstream primer N1NA-F: sequence is 5 '-GCAAAGCTTACCATGAATCCAAATCAGAAG-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, Hind III restriction enzyme site, KOZAK sequence, initiation codon and collochore; 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, with high-fidelity Taq enzyme, viral cDNA is carried out to pcr amplification, amplification NA fragment.
PCR product after HA genetic fragment, is used respectively Hind III and BamH I double digestion with expression vector pcDNA3 after agarose gel electrophoresis reclaims, and electrophoresis utilizes the HA fragment after T4DNA ligase cuts back to close enzyme to be connected with carrier after reclaiming again.NA fragment and green fluorescence expression vector are used after Hind III and BamH I double digestion, electrophoresis reclaims and connects, between the green fluorescent protein of this vector encoded and multiple clone site, there is a RES sequence (IRES), after the promoter of NA genetic fragment insertion vector, before IRES, when promoter startup is transcribed, can obtain the mRNA that a coding has NA, IRES and green fluorescent protein, final translation obtains independent NA and green fluorescent protein.
Connect product and transform respectively escherichia coli DH5a competent cell, coating Amp resistant panel.Bacterium colony, after PCR, extracts plasmid enzyme restriction and identifies and DNA sequencing, and obtaining the eukaryon expression plasmid that contains HA genetic fragment is pcDNA-H5HA, and obtaining the eukaryon expression plasmid that contains HA genetic fragment is pIRES-EGFP-N1NA.The bacterium that contains pcDNA-H5HA plasmid and pIRES-EGFP-N1NA plasmid, respectively after the LB overnight incubation of the ampicillin through containing 50 μ g/ml (Amp), with high-purity plasmid extraction kit, extract pcDNA-H5HA plasmid and pIRES-EGFP-N1NA plasmid, for follow-up transfectional cell and recombinant expressed experiment.
According to Lipofectamine 2000 kit explanations, by 2.5 μ g pcDNA-H5HA plasmids, 0.5 μ gpIRES-EGFP-N1NA plasmid and 10 μ l Lipofectamine 2000 transfection composites, 35mm diameter double dish of transfection contains Human Embryonic Kidney HEK-239 cell that stand density is about 80%-90% fusion rate, after transfection, 6h removes transfection composite, changes fresh complete medium (the DMEM nutrient culture media that contains 10% hyclone).
After transfection, HEK-239 cell gets final product instantaneous recombinant expressed HA, NA and green fluorescent protein, obtains the recombinant expressed influenza virus HA of instantaneous Green Fluorescent Protein and the cell of NA.
For further optimization, can screen to obtain and stablize the recombinant expressed HA of Green Fluorescent Protein and the cell of NA: after transfection 72h, transfectional cell piping and druming be disperseed, by in a new 35mm diameter double dish of 1/20 inoculation of cell suspension, supplement complete medium to 3ml, through spend the night adherent after, add G418 to concentration be 600 μ g/ml.After this replacing of every 3 to 5 days once contains the complete medium of 600 μ g/ml G418, the drug-resistant colonies that after approximately 3 weeks, visible screening occurs.Drug-resistant colonies, after trypsinization, is inoculated to 96 orifice plates by 10-20 cell/ml and is cultivated, add the complete medium that contains 600 μ g/ml G418 to be cultured to and grow to after individual layer, go down to posterity, obtain for detection of backup cultivate 96 orifice plates.The cell that detects 96 orifice plates of use is fixed to 5 minutes by 4% paraformaldehyde room temperature, after PBS rinsing, add the antibody of the fluorescently-labeled anti-H5 hypotype HA of Cy3 to carry out immunofluorescence detection, observe green fluorescence simultaneously, select red and green fluorescence positive signal by force, redness and the high hole of gfp positive cell ratio, cell is continued to the cloning and the immunofluorescence that repeat, approach 100% to positive cell ratio, can obtain stablizing the cell of Green Fluorescent Protein recombinant expressed this strain HA and NA.
Green Fluorescent Protein is instantaneous or stablize the cell of recombinant expressed this strain HA and NA, through adherent or suspend and cultivate after, with the PBS that contains 2%BSA, cell piping and druming is disperseed, cell suspension is carried out centrifugal, remove after supernatant with the PBS that contains 2%BSA by 10
5individual/ml cell density carries out resuspended.Remove supernatant, with isopyknic PBS is resuspended, centrifugal, wash 2 times, again add isopyknic PBS, add isopyknic 10% cold formaldehyde, in 4 ℃, fix 2 hours.Through the mode centrifugal, PBS is resuspended, carry out 3 washings.Finally by cell precipitation, by 10
5the concentration of individual/ml is resuspended in the PBS damping fluid that contains 0.2% Nepal's gold ester, 2%BSA, 20% glycerine, 0.5% heparin, can stably preserve for a long time, obtain the recombinant expressed HA of Green Fluorescent Protein after processing and the eukaryotic of NA antigen.
By the eukaryotic cell suspension of the recombinant expressed HA of this Green Fluorescent Protein and NA antigen 25 μ l and sample to be checked 25 μ l, in micro-agglutination plate, to mix, room temperature is placed after 60 minutes, at fluorescence microscopy Microscopic observation.As there is not aggegation, and the cell that sends green fluorescence can be deposited in agglutination plate bottom, and under mirror, visible fluorescence cell aggregation is in central authorities; As there is aggegation, and the cell that sends green fluorescence floats on a liquid with cotton-shaped, and under mirror, visible fluorescence cell disperses in the visual field, cell and iuntercellular generation agglomerate.Utilize this feature, also can carry out imaging with fluorescent microscope, utilize image analysis software, by setting up fluorescence radiation area whether to realize software interpretation aggegation.During sample generation aggegation to be checked, illustrate in sample to be checked and contain with this strain HA and/or NA and there is reactive influenza virus HA and/or NA antibody.
The recombinant expressed influenza virus HA of embodiment 2 fluorescein CFSE (carbox fluorescenceindiacetate succinimidyl ester) marks and the eukaryotic method of NA
It is example that the present embodiment be take 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 separated strain H5N1 subtype influenza virus obtaining in the chicken in 2005 Nian Guangdong Province, 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.To after this strain virus propagation, use Viral RNA Miniprep Kit to extract viral RNA, with Uni-12 primer (5 '-AGCAAAAGCAGG-3 ') and SuperScript III or M-MLV reverse transcriptase, carry out reverse transcription, obtain viral cDNA.According to the gene order of this strain HA, design a pair of primer for HA full-length gene clone, its primer sequence is specific as follows: upstream primer H5HA-F, sequence is 5 '-GCAAAGCTTACCATGGAGAAAATAGTACTTCTTC-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, Hind III restriction enzyme site, KOZAK sequence, initiation codon and collochore; Downstream primer H5HA-R, sequence is 5 '-CTCGGATCCTTAAATGCAAATTCTGCATTGTAAG-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, BamH I restriction enzyme site, terminator codon and collochore.Utilize this to primer, with high-fidelity Taq enzyme, viral cDNA is carried out to pcr amplification, amplification HA fragment.
According to the gene order of this strain NA, design a pair of primer for NA full-length gene clone, its primer sequence is specific as follows: upstream primer N1NA-F: sequence is 5 '-GCAAAGCTTACCATGAATCCAAATCAGAAG-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, Hind III restriction enzyme site, KOZAK sequence, initiation codon and collochore; 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, with high-fidelity Taq enzyme, viral cDNA is carried out to pcr amplification, amplification NA fragment.
PCR product is after agarose gel electrophoresis reclaims after HA and NA genetic fragment, use respectively Hind III and BamH I double digestion with expression vector pcDNA3, electrophoresis utilizes after reclaiming that HA fragment after T4DNA ligase cuts back to close enzyme is connected with carrier, NA fragment is connected with carrier again, transform respectively escherichia coli DH5a competent cell, coating Amp resistant panel.Bacterium colony, after PCR, extracts plasmid enzyme restriction and identifies and DNA sequencing, and obtaining the eukaryon expression plasmid that contains HA genetic fragment is pcDNA-H5HA, and obtaining the eukaryon expression plasmid that contains 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), with high-purity plasmid extraction kit, extract pcDNA-H5HA plasmid and pcDNA-N1NA plasmid, for follow-up transfectional cell and recombinant expressed experiment.
According to Lipofectamine 2000 kit explanations, by 2.5 μ g pcDNA-H5HA plasmids, 0.5 μ gpcDNA-N1NA plasmid and 10 μ l Lipofectamine 2000 transfection composites, 35mm diameter double dish of transfection contains Human Embryonic Kidney HEK-239 cell that stand density is about 80%-90% fusion rate, after transfection, 6h removes transfection composite, changes fresh complete medium (the DMEM nutrient culture media that contains 10% hyclone).
After transfection, HEK-239 cell gets final product instantaneous recombinant expressed HA and NA, obtains the cell of instantaneous recombinant expressed influenza virus HA and NA.
For further optimization, can screen and obtain the cell of stablizing recombinant expressed HA and NA: after transfection 72h, transfectional cell piping and druming be disperseed, by in a new 35mm diameter double dish of 1/20 inoculation of cell suspension, supplement complete medium to 3ml, through spend the night adherent after, add G418 to concentration be 600 μ g/ml.After this replacing of every 3 to 5 days once contains the complete medium of 600 μ g/ml G418, the drug-resistant colonies that after approximately 3 weeks, visible screening occurs.Drug-resistant colonies, after trypsinization, is inoculated to 96 orifice plates by 10-20 cell/ml and is cultivated, add the complete medium that contains 600 μ g/ml G418 to be cultured to and grow to after individual layer, go down to posterity, obtain for detection of backup cultivate 96 orifice plates.The cell that detects 96 orifice plates of use is fixed to 5 minutes by 4% paraformaldehyde room temperature, after 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 to carry out immunofluorescence detection, select red and green fluorescence positive signal by force, redness and the high hole of gfp positive cell ratio, cell is continued to the cloning and the immunofluorescence that repeat, approach 100% to positive cell ratio, can obtain stablizing the cell of recombinant expressed this strain HA and NA.
Instantaneous or stablize the cell of recombinant expressed this strain HA and NA, through adherent or suspend and cultivate after, with the PBS that contains 2%BSA, cell piping and druming is disperseed, cell suspension is carried out centrifugal, remove after supernatant with the PBS that contains 0.1%BSA by 10
7individual/ml cell density carries out resuspended.In every ml cell suspension, the CFSE mother liquor that adds the 5mmol/L of 2 μ l, place 10 minutes for 37 ℃, the PBS that adds again the precooling of 5ml ice places 5 minutes in mixture of ice and water, centrifugal removal supernatant, with isopyknic PBS is resuspended, centrifugal, wash 2 times, again add isopyknic PBS, add isopyknic 10% cold formaldehyde, in 4 ℃, fix 2 hours.Through the mode centrifugal, PBS is resuspended, carry out 3 washings.Finally by cell precipitation, by the concentration of 105/ml, be resuspended in the PBS damping fluid that contains 0.2% Nepal's gold ester, 2%BSA, 20% glycerine, 0.5% heparin, can stably preserve for a long time, obtain the recombinant expressed HA of fluorescein CFSE mark and the eukaryotic of NA antigen.
By the eukaryotic cell suspension of the recombinant expressed HA of this Green Fluorescent Protein and NA antigen 25 μ l and sample to be checked 25 μ l, in micro-agglutination plate, to mix, room temperature is placed after 60 minutes, at fluorescence microscopy Microscopic observation.As there is not aggegation, and the cell that sends green fluorescence can be deposited in agglutination plate bottom, and under mirror, visible fluorescence cell aggregation is in central authorities; As there is aggegation, and the cell that sends green fluorescence floats on a liquid with cotton-shaped, and under mirror, visible fluorescence cell disperses in the visual field, cell and iuntercellular generation agglomerate.Utilize this feature, also can carry out imaging with fluorescent microscope, utilize image analysis software, by setting up fluorescence radiation area whether to realize software interpretation aggegation.During sample generation aggegation to be checked, illustrate in sample to be checked and contain with this strain HA and/or NA and there is reactive influenza virus HA and/or NA antibody.
The eukaryotic method of embodiment 3 green fluorescent proteins and the red fluorescent protein difference independent recombinant expressed influenza virus HA of mark and NA
It is example that the present embodiment be take the strain A/Chicken/Guangdong/1/2005 (H5N1) of a strain H5 type, carries out the preparation of recombinant expressed influenza virus hemagglutinin HA eukaryotic expression cell.A/Chicken/Guangdong/1/2005 (H5N1) is the separated strain H5N1 subtype influenza virus obtaining in the chicken in 2005 Nian Guangdong Province, 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.To after this strain virus propagation, use Viral RNA Miniprep Kit to extract viral RNA, with Uni-12 primer (5 '-AGCAAAAGCAGG-3 ') and SuperScript III or M-MLV reverse transcriptase, carry out reverse transcription, obtain viral cDNA.According to the gene order of this strain HA, design a pair of primer for HA full-length gene clone, its primer sequence is specific as follows: upstream primer H5HA-F, sequence is 5 '-GCAAAGCTTACCATGGAGAAAATAGTACTTCTTC-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, Hind III restriction enzyme site, KOZAK sequence, initiation codon and collochore; Downstream primer H5HA-R, sequence is 5 '-CTCGGATCCTTAAATGCAAATTCTGCATTGTAAG-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, BamH I restriction enzyme site, terminator codon and collochore.Utilize this to primer, with high-fidelity Taq enzyme, viral cDNA is carried out to pcr amplification, amplification HA fragment.
According to the gene order of this strain NA, design a pair of primer for NA full-length gene clone, its primer sequence is specific as follows: upstream primer N1NA-F: sequence is 5 '-GCAAAGCTTACCATGAATCCAAATCAGAAG-3 ', from 5 ' end, is followed successively by 3 protectiveness bases, Hind III restriction enzyme site, KOZAK sequence, initiation codon and collochore; 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, with high-fidelity Taq enzyme, viral cDNA is carried out to pcr amplification, amplification NA fragment.
PCR product is after agarose gel electrophoresis reclaims after HA genetic fragment, use respectively Hind III and BamH I double digestion with red fluorescence expression vector, electrophoresis utilizes the HA fragment after T4DNA ligase cuts back to close enzyme to be connected with carrier after reclaiming again.This red fluorescence expression vector, between the red fluorescent protein of encoding and multiple clone site, there is a RES sequence (IRES), after the promoter of HA genetic fragment insertion vector, before IRES, when promoter startup is transcribed, can obtain the mRNA that a coding has HA, IRES and red fluorescent protein, final translation obtains independent HA and red fluorescent protein.NA fragment and green fluorescence expression vector are used after Hind III and BamH I double digestion, electrophoresis reclaims and connects, between the green fluorescent protein of this vector encoded and multiple clone site, there is a RES sequence (IRES), after the promoter of NA genetic fragment insertion vector, before IRES, when promoter startup is transcribed, can obtain the mRNA that a coding has NA, IRES and green fluorescent protein, final translation obtains independent NA and green fluorescent protein.
Connect product and transform respectively escherichia coli DH5a competent cell, coating Amp resistant panel.Bacterium colony, after PCR, extracts plasmid enzyme restriction and identifies and DNA sequencing, and obtaining the eukaryon expression plasmid that contains HA genetic fragment is pIRES-Red-H5HA, and obtaining the eukaryon expression plasmid that contains HA genetic fragment is pIRES-EGFP-N1NA.The bacterium that contains pIRES-Red-H5HA plasmid and pIRES-EGFP-N1NA plasmid, respectively after the LB overnight incubation of the ampicillin through containing 50 μ g/ml (Amp), with high-purity plasmid extraction kit, extract pIRES-Red-H5HA plasmid and pIRES-EGFP-N1NA plasmid, for follow-up transfectional cell and recombinant expressed experiment.
According to Lipofectamine 2000 kit explanations, by 3 μ g pIRES-Red-H5HA plasmids and 10 μ lLipofectamine 2000 transfection composites, 35mm diameter double dish of transfection contains Human Embryonic Kidney HEK-239 cell that stand density is about 80%-90% fusion rate, after transfection, 6h removes transfection composite, changes fresh complete medium (the DMEM nutrient culture media that contains 10% hyclone).
After transfection, HEK-239 cell gets final product instantaneous recombinant expressed HA, obtains the cell of the instantaneous recombinant expressed influenza virus HA of red fluorescent protein mark.
For further optimization, can screen and obtain the cell of stablizing recombinant expressed HA: after transfection 72h, transfectional cell piping and druming be disperseed, by in a new 35mm diameter double dish of 1/20 inoculation of cell suspension, supplement complete medium to 3ml, through spend the night adherent after, add G418 to concentration be 600 μ g/ml.After this replacing of every 3 to 5 days once contains the complete medium of 600 μ g/ml G418, the drug-resistant colonies that after approximately 3 weeks, visible screening occurs.By drug-resistant colonies after trypsinization, by 10-20 cell/ml, inoculating 96 orifice plates cultivates, adding the complete medium that contains 600 μ g/ml G418 to be cultured to grows to after individual layer, the red fluorescence of observation of cell under fluorescent microscope, cell is continued to the cloning repeating, approach 100% to red fluorescence positive cell ratio, can obtain the cell of recombinant expressed this strain of stablizing of red fluorescent protein mark HA.
According to Lipofectamine 2000 kit explanations, by 3 μ g pIRES-EGFP-N1NA plasmids and 10 μ lLipofectamine 2000 transfection composites, 35mm diameter double dish of transfection contains Human Embryonic Kidney HEK-239 cell that stand density is about 80%-90% fusion rate, after transfection, 6h removes transfection composite, changes fresh complete medium (the DMEM nutrient culture media that contains 10% hyclone).
After transfection, HEK-239 cell gets final product instantaneous recombinant expressed NA, obtains the cell of the instantaneous recombinant expressed influenza virus NA of Green Fluorescent Protein.
For further optimization, can screen and obtain the cell of stablizing recombinant expressed NA: after transfection 72h, transfectional cell piping and druming be disperseed, by in a new 35mm diameter double dish of 1/20 inoculation of cell suspension, supplement complete medium to 3ml, through spend the night adherent after, add G418 to concentration be 600 μ g/ml.After this replacing of every 3 to 5 days once contains the complete medium of 600 μ g/ml G418, the drug-resistant colonies that after approximately 3 weeks, visible screening occurs.By drug-resistant colonies after trypsinization, by 10-20 cell/ml, inoculating 96 orifice plates cultivates, adding the complete medium that contains 600 μ g/ml G418 to be cultured to grows to after individual layer, fluorescence microscopy Microscopic observation green fluorescence, select the hole that fluorescence positive signal is strong, fluorescencepositive cell ratio is high, cloning and immunofluorescence that cell continue is repeated, approach 100% to positive cell ratio, can obtain the cell of recombinant expressed this strain of stablizing of Green Fluorescent Protein NA.
Red fluorescent protein mark instantaneous or stablize the cell of recombinant expressed this strain HA, after cultivating, with the gentle vitellophag of low concentration pancreatin (0.05%) approximately 5 minutes, add the nutrient culture media cessation reaction that contains hyclone, cell suspension is carried out centrifugal, after removal supernatant, use the PBS that contains 2%BSA to carry out resuspended, obtain the cell suspension after pancreatin is processed.For preventing that cell that HA may cause is from aggegation, in cell suspension after pancreatin is processed, add neuraminidase (to claim again neuraminidase, Neuraminidase) process, eliminate the effect of recombinant expressed HA and cell autoreceptor, avoid cell autoagglutination.That is, cell suspension, after centrifugal, with 50mM sodium citrate (pH 6.0) damping fluid, is pressed to 10 by cell
7individual/ml carries out resuspended, adds the neuraminidase of 200 units in every ml cell suspension, centrifugal process 30 minutes under 37 ℃ of conditions after, removes supernatant, with isopyknic PBS is resuspended, centrifugal, washs 2 times, again adds isopyknic PBS.The above-mentioned cell suspension of processing through pancreatin and neuraminidase, adds isopyknic 10% cold formaldehyde, in 4 ℃, fixes 2 hours.Through the mode centrifugal, PBS is resuspended, carry out 3 washings.Finally by cell precipitation, by 10
5the concentration of individual/ml is resuspended in the PBS damping fluid that contains 0.2% Nepal's gold ester, 2%BSA, 20% glycerine, 0.5% heparin, can stably preserve for a long time, has obtained the eukaryotic of the recombinant expressed HA antigen after processing.
Green Fluorescent Protein instantaneous or stablize the cell of recombinant expressed this strain NA, through adherent or suspend and cultivate after, with the PBS that contains 2%BSA, cell piping and druming is disperseed, cell suspension is carried out centrifugal, remove after supernatant with the PBS that contains 2%BSA by 10
7individual/ml cell density carries out resuspended.Remove supernatant, with isopyknic PBS is resuspended, centrifugal, wash 2 times, again add isopyknic PBS, add isopyknic 10% cold formaldehyde, in 4 ℃, fix 2 hours.Through the mode centrifugal, PBS is resuspended, carry out 3 washings.Finally by cell precipitation, by 10
5the concentration of individual/ml is resuspended in the PBS damping fluid that contains 0.2% Nepal's gold ester, 2%BSA, 20% glycerine, 0.5% heparin, can stably preserve for a long time, has obtained the eukaryotic of the recombinant expressed NA antigen after processing.
Above-mentioned green fluorescent protein and the red fluorescent protein respectively independent recombinant expressed HA of mark and the eukaryotic of NA are 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 in detection effect.
By the eukaryotic suspension of the independent recombinant expressed HA of green fluorescent protein or NA and the red fluorescent protein difference independent recombinant expressed HA of mark or the eukaryotic suspension of NA 25 μ l and sample to be checked 25 μ l, in micro-agglutination plate, mix, room temperature is placed after 60 minutes, at fluorescence microscopy Microscopic observation.As there is not aggegation, and the cell that sends green fluorescence can be deposited in agglutination plate bottom, and under mirror, visible fluorescence cell aggregation is in central authorities; As there is aggegation, and the cell that sends green fluorescence floats on a liquid with cotton-shaped, and under mirror, visible fluorescence cell disperses in the visual field, cell and iuntercellular generation agglomerate.Utilize this feature, also can carry out imaging with fluorescent microscope, utilize image analysis software, by setting up fluorescence radiation area whether to realize software interpretation aggegation.During sample generation aggegation to be checked, illustrate in sample to be checked and contain with this strain HA and/or NA and there is reactive influenza virus HA and/or NA antibody.
By the eukaryotic suspension (being made as A) of the recombinant expressed HA antigen of red fluorescent protein mark, the eukaryotic suspension (being made as B) of the recombinant expressed NA antigen of Green Fluorescent Protein, the recombinant expressed HA of Green Fluorescent Protein and the eukaryotic suspension of NA antigen (or green fluorescent protein and the red fluorescent protein difference single expression HA of mark and the eukaryotic mixing suspension of NA, be made as C), respectively adding 25 μ l is added in clean micro-agglutination plate, then sample (the serum to be checked that respectively adds 25 μ l, bronchial perfusate, blood plasma, tissue fluid etc.) to 3 kinds of different cell suspensions, mix, room temperature was placed after 60 minutes, at fluorescence microscopy Microscopic observation.As there is not aggegation, and the cell that sends green fluorescence can be deposited in agglutination plate bottom, and under mirror, visible fluorescence cell aggregation is in central authorities; As there is aggegation, and the cell that sends green fluorescence floats on a liquid with cotton-shaped, and under mirror, visible fluorescence cell disperses in the visual field, cell and iuntercellular generation agglomerate.Utilize this feature, also can carry out imaging with fluorescent microscope, utilize image analysis software, by setting up fluorescence radiation area whether to realize software interpretation aggegation.During sample generation aggegation to be checked, illustrate in sample to be checked and contain with this strain HA and/or NA and there is reactive influenza virus HA and/or NA antibody.
Set up positive serum and negative serum contrast, as control sample meets expected results, sample to be checked occurs, to illustrate in sample to be checked and do not contain with this strain HA and NA and have the detectable concentration that reactive influenza virus HA and NA antibody or antibody concentration can reach lower than the method all not during aggegation with above-mentioned cell suspension.Set up positive serum and negative serum contrast, as control sample meets expected results, when sample to be checked occurs all aggegation to occur with above-mentioned cell suspension, illustrate in sample to be checked and contain with this strain HA and NA and there is reactive influenza virus HA and NA antibody.
Set up positive serum and negative serum contrast, as control sample meets expected results, when sample to be checked occurs all aggegation to occur with cell suspension A and cell suspension C, and while there is not aggegation with cell suspension B, illustrate in sample to be checked and contain with this strain HA and there is reactive influenza virus HA antibody not there is and do not contain with this strain NA the detectable concentration that reactive influenza virus NA antibody or antibody concentration can reach lower than the method.
Set up positive serum and negative serum contrast, as control sample meets expected results, when sample to be checked occurs all aggegation to occur with cell suspension B and cell suspension C, and while there is not aggegation with cell suspension A, illustrate in sample to be checked and contain with this strain NA and there is reactive influenza virus NA antibody not there is and do not contain with this strain HA the detectable concentration that reactive influenza virus HA antibody or antibody concentration can reach lower than the method.
During sample generation aggegation to be checked, illustrate in sample to be checked and contain with this strain HA and/or NA and there is reactive influenza virus HA and/or NA antibody.Now can further carry out relative quantitative assay,, sample 1 to be checked is carried out to 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, the high dilution of sample that obtains occurring aggegation, is made as d1.Same method can obtain the high dilution d2 of the agglutinating reaction positive of sample 2 to be checked.Concentration difference and relative scale with influenza virus HA and/or NA antibody in the different samples to be checked of this lateral comparison.
A kind of fluorescence few cells aggegation method sequence table that detects Antibody of Influenza
SEQUENCE LISTING
<110> Medical College of Shantou University
<120> fluorescence few cells aggegation method that detects Antibody of Influenza
<130>
<160>5
<170>PatentIn version 3.2
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agcaaaagca gg 12
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<213> artificial sequence
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gcaaagctta ccatggagaa aatacttctt c 31
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<212>DNA
<213> artificial sequence
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ctcggatcct taaatgcaaa ttctgcattg taag 34
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gcaaagctta ccatgaatcc aaatcagaag 30
<210>5
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<212>DNA
<213> artificial sequence
<400>5
ctcggatccc tacttgtcaa tggtgaatg 29
Claims (2)
1. a fluorescence few cells aggegation method that detects Antibody of Influenza, it is characterized in that described method is by transfecting eukaryotic cells after influenza virus hemagglutinin gene and Neuraminidase Gene clone, make the eukaryotic recombinant expressed influenza virus hemagglutinin in surface and neuraminic acid zymoprotein, restructuring express cell is carried out to fluorescence labeling, the recombinant expressed hemagglutinin of this cell surface and neuraminic acid zymoprotein are combined with flu virus hemagglutinin antibody and the neuraminic acid enzyme antibody of testing sample, there is agglutinating reaction, fluoroscopic examination is analyzed, according to fluorescence area, whether increase to judge in testing sample, whether have Antibody of Influenza, described eukaryotic is human embryo kidney (HEK) HEK-239 cell.
2. the fluorescence few cells aggegation method of detection Antibody of Influenza 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, or the eukaryotic of recombinant expressed influenza virus hemagglutinin mixes with the eukaryotic of recombinant expressed neuraminidase respectively.
3. the fluorescence few cells aggegation method of detection Antibody of Influenza according to claim 1, it is characterized in that described recombinant expressed be transient expression or stably express.
4. the fluorescence few cells aggegation method of detection Antibody of Influenza 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 epitope of influenza virus hemagglutinin and neuraminidase.
5. the fluorescence few cells aggegation method of detection Antibody of Influenza according to claim 1, is characterized in that the hemagglutinin of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 hypotype, influenza B virus or influenza virus C that the recombinant expressed hemagglutinin in described eukaryotic surface is influenza A virus.
6. the fluorescence few cells aggegation method of detection Antibody of Influenza according to claim 1, is characterized in that the neuraminidase of N1, N2, N3, N4, N5, N6, N7, N8, N9 hypotype, influenza B virus or influenza virus C that the recombinant expressed neuraminic acid zymoprotein in described eukaryotic surface is influenza A virus.
7. the fluorescence few cells aggegation method of detection Antibody of Influenza according to claim 1, is characterized in that described flu virus hemagglutinin antibody and neuraminic acid enzyme antibody are one or more the potpourri in IgG, IgM, IgA, IgE, IgD.
8. the fluorescence few cells aggegation method of detection Antibody of Influenza according to claim 1, is characterized in that described fluorescence labeling is that fluorescein, fluorescin or other can send the material of fluorescence.
9. the fluorescence few cells aggegation method of detection Antibody of Influenza according to claim 1, is characterized in that it is fluorescent microscope, fluorophotometer or multi-functional microwell plate detector that equipment used is analyzed in described fluoroscopic examination.
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