CN102023212B - Rapid detection method of influenza virus neuraminidase antibody - Google Patents
Rapid detection method of influenza virus neuraminidase antibody Download PDFInfo
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Abstract
The invention belongs to the technical field of biology and particularly relates to a rapid detection method of an influenza virus neuraminidase antibody. The detection method comprises the following steps of: coning the influenza virus neuraminidase gene and then transfecting eukaryotic cells so that influenza virus neuraminidase protein is in recombination expression on the surface of eukaryotic cells, combining with the influenza virus neuraminidase antibody in a sample to be detected, performing agglutination reaction and judging whether the influenza virus neuraminidase antibody exists in the sample to be detected according to whether the agglutination reaction happens or not. The method is simple, rapid, stable and highly sensitive, and has wide application value.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method for quick of influenza neuraminidase antibody.
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 influenza neuraminidase 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.
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.
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.And said method can only be for detection and the Serotype Identification of influenza virus hemagglutinin (HA) antibody.
The at present detection to NA serotype antibody, the method that mainly contains neuraminidase inhibition of enzyme activity detects, and ELISA method and latex agglutination experimental technique detect.Wherein, ELISA method is to utilize recombinant expressed and influenza virus 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 NA antigen of different serotypes is all 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 (suppressing to detect as ELISA method and NA are active), and the reaction time is short, be applicable to Site Detection, but still comes with some shortcomings.Latex agglutination experimental technique is on inertia latex particle surface by the influenza virus NA albumen coupling of recombinant expressed and purifying, utilize agglutinating reaction to detect the corresponding antibody of influenza virus, this needs the influenza virus 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 the influenza virus NA antibody of different serotypes, just need to carry out recombinant expressed and purifying to the NA of different strains, the same with ELISA method, have that workload is large, technological requirement is high, batch between the problem such as poor stability.And the method for neuraminidase inhibition of enzyme activity detects, need the NA after purifying, and the essential biologically active that keeps NA, it is the enzymatic activity that NA digests specific sugar chain, sample to be checked is added, as contain corresponding NA antibody, its NA is active will be reduced, by specific substrate, detect, can judge and in sample to be checked, whether contain NA antibody, the method needs more complicated technique to obtain the NA recombinant protein that maintains enzymatic activity of different serotypes, need the particular procedure purifying substrate (2 ' of (as containing fluorophor and fluorescent quenching group double-tagging) simultaneously, 3 '-sialic acid or 2 ', 6 '-sialic acid acceptor or derivant etc.), need to detect by fluorospectrophotometer or miscellaneous equipment in addition.
Owing to detecting Antibody of Influenza, whether people or this animal are infected to the influenza virus of (comprise early infection, subclinical infection or infect then recover etc.) this particular serotype or inoculated influenza virus vaccine or evaluated that it is significant to the resistibility of particular serotype influenza virus etc., and, especially lack the method that is suitable for fast Site Detection for NA antibody at present.And the current detection meanss such as the experiment of NA inhibition of enzyme activity, ELISA and latex agglutination, 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 influenza virus NA antibody detection method easy, quick, that technique is relatively simple and easy to control is with a wide range of applications.
Summary of the invention
The object of the invention is to according to detecting in prior art the shortcomings such as the workload existing in influenza neuraminidase antibody is large, length consuming time, provide a kind of fast, the simple method for quick of the influenza neuraminidase antibody of the eukaryotic agglutinating reaction based on ethnic expression of influenza virus neuraminidase.
The object of the invention is achieved by the following technical programs:
A kind of method for quick of influenza neuraminidase antibody, described detection method is by transfecting eukaryotic cells after Neuraminidase of Influenza Virus A clone, make the recombinant expressed influenza neuraminidase albumen in eukaryotic surface, as agglutinating reaction matrix, can be combined by the influenza neuraminidase antibody in sample to be checked, there is agglutinating reaction, according to whether there is agglutinating reaction, can judge in sample to be checked, whether there is influenza neuraminidase antibody.
As a kind of preferred version, described influenza neuraminidase is the neuraminic acid zymoprotein of complete total length, an or part for influenza neuraminidase, or the epitope of influenza neuraminidase, or the partial amino-acid in influenza neuraminidase albumen total length or partial peptide section is suddenlyd change or modified.
Influenza virus comprises influenza A virus, influenza B virus and influenza virus C, wherein the NA of influenza A virus can be divided into 9 hypotypes, meanwhile, and due to sudden change and the evolution of influenza virus NA, even the NA of same hypotype, 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 NA of recombinant expressed this particular serotype strain and sample to be checked are carried out to aggegation experiment.Above-mentioned influenza virus NA, refer to N1, N2, N3, N4, N5, N6, N7, N8, the N9 hypotype of influenza A virus, and the neuraminidase of influenza B virus, influenza virus C, can be according to the NA that need to use a certain particular serotype of testing goal or its part.
The Antibody of Influenza of carrying out in this way more extensive serotype detects, can carry out antigenicity analysis according to the NA of detected object serotype strain, select an antigenicity part conservative or the NA albumen that antigenic cross property is strong to carry out recombinant expressed eukaryotic structure, utilize agglutinating reaction to detect the Antibody of Influenza of more extensive serotype.Above-mentioned influenza virus NA, can be a certain influenza virus strain NA or wherein a part of, also can be the NA of various flows Influenza Virus strain or wherein splicing or the mixing of a part or different N A, also can be that infected by influenza NA suddenlys change and modifies, also can be artificial synthetic natural non-existent sequence, but its core is recombination expression product, there is the antigenicity of influenza virus NA.
The eukaryotic of above-mentioned recombinant expressed influenza neuraminidase (NA), refers to and has the genetic fragment of influenza virus NA to import eukaryotic coding, at 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 NA is positioned at cell membrane or cell membrane surface.Wherein, eukaryotic preferred mammal cell and insect cell, its recombinant expressed influenza neuraminidase 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 NA of preferred mammal cell.Recombinant expressed can be transient expression, can be also stably express.It can be DNA that coding has the genetic fragment of influenza virus 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 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 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 NA is entered to eukaryotic is recombinant expressed to obtain.The eukaryotic of above-mentioned recombinant expressed influenza neuraminidase (NA), its core is to obtain the eukaryotic that has influenza virus NA at cell surface expression, its objective is the antigenicity of utilizing expressed NA, by aggegation, test, detect Antibody of Influenza.In one embodiment of the invention, announced the eukaryotic method of recombinant expressed influenza neuraminidase (NA).
The eukaryotic of above-mentioned recombinant expressed influenza neuraminidase (NA), as the particulate antigen matrix for agglutinating reaction, it can be the eukaryotic of natural recombinant expressed influenza neuraminidase, or through pancreatin processing, mechanical dispersion, enzymolysis disperse, the technique such as fixing agent is fixed, stabilizer treatment, antiseptic are processed simultaneously or the eukaryotic of the recombinant expressed influenza neuraminidase of selectivity after processing, better to make " agglutinating reaction matrix " to preserve, prevent self-solidifying, to increase sensitivity and stability that agglutinating reaction detects.In one embodiment of the invention, announced the eukaryotic disposal route of recombinant expressed influenza neuraminidase (NA), the sensitivity and the stability that to improve agglutinating reaction, detect.
As a kind of preferred version, described sample to be checked is behaved or animal body fluid, as serum, bronchial perfusate, blood plasma or tissue fluid.
As a kind of preferred version, described influenza neuraminidase antibody is one or more the potpourri in IgG, IgM, IgA, IgE, IgD.
As a kind of preferred version, described recombinant expressed be transient expression or stably express.
Compared with prior art, the present invention has following beneficial effect:
The present invention be take the cell of recombinant expressed influenza virus NA as carrier, utilizes agglutinating reaction, detects the antibody that whether has respective streams Influenza Virus in sample to be checked.Utilize 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 NA in cell surface, after hydroformylation is fixing, this cell becomes the sensitization particle that contains NA antigen in surface, after sample mix to be checked, as the NA antibody that contains influenza virus in sample to be checked, can there is at several minutes agglutinating reaction.The method does not relate to and has infective virion; Compare ELISA and NA inhibition of enzyme activity and test required a few hours to a couple of days, the reaction of the cell agglutination of recombinant expressed NA only needs several minutes, and the reaction time is quick; Utilize universal primer and carrier to clone and transfection expression the NA gene of different serotypes, can obtain the sensitized cell particle of different N Staphylococal Protein A, can to different N A serotype influenza virus, detect more widely.
Embodiment
Below in conjunction with embodiment, further explain the present invention, but embodiment does not limit in any form to the present invention.
The eukaryotic preparation of the recombinant expressed influenza neuraminidase of embodiment 1 (NA)
It is example that the present embodiment be take the strain A/Chicken/Guangdong/1/2005 (H5N1) of a strain H5N1 hypotype, carries out the preparation of recombinant expressed influenza neuraminidase NA 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, and the gene order of its NA can obtain on public database GenBank, 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 NA, design a pair of primer for NA full-length gene clone, its primer sequence is specific as follows: upstream primer N 1NA-F: sequence is 5 '-GTAAAGCTTACCATGAATCCAAATCAGAAG-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 '-CACGGATCCCTACTTGTCAATGGTGAATG-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 NA genetic fragment, use respectively Hind III and BamH I double digestion with expression vector pcDNA3, electrophoresis utilizes the NA fragment after T4DNA ligase cuts back to close enzyme to be connected with carrier after reclaiming 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 NA genetic fragment is pcDNA-N1NA.
The bacterium that contains pcDNA-N1NA plasmid, after the LB overnight incubation of the ampicillin through containing 50 μ g/ml (Amp), extracts pcDNA-N1NA plasmid with high-purity plasmid extraction kit.According to Lipofectamine 2000 kit explanations, by 3 μ g pcDNA-N1NA plasmids and 10 μ l Lipofectamine2000 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 instantaneous recombinant expressed influenza virus NA.
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.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, to be that primary antibodie, Cy3 fluorescently-labeled two is anti-carry out immunofluorescence detection to the antibody that adds anti-N1 hypotype NA, select the hole that fluorescence positive signal is strong, fluorescencepositive cell ratio is high, 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 NA.
The eukaryotic processing of the recombinant expressed influenza neuraminidase of embodiment 2 (NA)
Instantaneous or stablize the cell of recombinant expressed this strain NA, after cultivating, with EDETATE SODIUM (0.02%) room temperature is gentle, process cell approximately 2 minutes, 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 EDTA processes.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 107/ml, be resuspended in the PBS damping fluid that contains 0.2% Nepal's gold ester, 2%BSA, 15% glycerine, 0.5% heparin, can stably preserve for a long time, obtain the eukaryotic of the recombinant expressed NA antigen after processing.
Embodiment 3 is used the eukaryotic of recombinant expressed influenza neuraminidase to carry out agglutinating reaction and detects Antibody of Influenza
1 of the eukaryotic suspension of the recombinant expressed NA antigen after the processing in embodiment 2 (approximately 50 μ l) is added on clean microslide, then the sample to be checked (serum, bronchial perfusate, blood plasma, tissue fluid etc.) that adds 50 μ l, rock gently microslide all around or use micropipette tip to be mixed, within room temperature 1-3 minute, observe whether occur cell agglutination.
Set up positive serum and negative serum contrast, as control sample meets expected results, when not aggegation occurs sample to be checked, illustrate in sample to be checked, not contain with this strain NA to there is the detectable concentration that reactive influenza virus NA 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 NA and there is reactive influenza virus NA antibody.Now can further carry out relative quantitative assay, that is, sample 1 to be checked is carried out to continuous doubling dilution, then respectively with embodiment 2 in processing after the eukaryotic suspension of recombinant expressed NA antigen carry out agglutinating reaction, the high dilution 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 NA antibody in the different samples to be checked of this lateral comparison.
A kind of method for quick sequence table of influenza neuraminidase antibody
SEQUENCE LISTING
<110> Medical College of Shantou University
The method for quick of a <120> influenza neuraminidase antibody
<130>
<160>3
<170>PatentIn version 3.2
<210>1
<211>12
<212>DNA
<213> artificial sequence
<400>1
agcaaaagca gg 12
<210>2
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<212>DNA
<213> artificial sequence
<400>2
gtaaagctta ccatgaatcc aaatcagaag 30
<210>3
<211>29
<212>DNA
<213> artificial sequence
<400>3
cacggatccc tacttgtcaa tggtgaatg 29
Claims (2)
1. the method for quick of an influenza neuraminidase antibody, it is characterized in that described detection method is by transfecting eukaryotic cells after Neuraminidase of Influenza Virus A clone, make the recombinant expressed influenza neuraminidase albumen in eukaryotic surface, as agglutinating reaction matrix, can be combined by the influenza neuraminidase antibody in sample to be checked, there is agglutinating reaction, according to whether there is agglutinating reaction, can judge in sample to be checked, whether there is influenza neuraminidase antibody; Described eukaryotic is human embryo kidney (HEK) HEK-239 cell.
2. the method for quick of influenza neuraminidase antibody according to claim 1, it is characterized in that described influenza neuraminidase is the neuraminic acid zymoprotein of complete total length, an or part for influenza neuraminidase, or the epitope of influenza neuraminidase, or the partial amino-acid in influenza neuraminidase albumen total length or partial peptide section is suddenlyd change or modified.
3. the method for quick of influenza neuraminidase antibody according to claim 1, is characterized in that described influenza neuraminidase 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.
4. the method for quick of influenza neuraminidase antibody according to claim 1, it is characterized in that described recombinant expressed influenza neuraminidase is independent influenza neuraminidase albumen or partial peptide section, or carry out amalgamation and expression or common independent expression with other albumen, polypeptide.
5. the method for quick of influenza neuraminidase antibody according to claim 1, it is characterized in that described agglutinating reaction matrix is the eukaryotic of natural recombinant expressed influenza neuraminidase, or through pancreatin processing, mechanical dispersion, enzymolysis disperse, fixing agent is fixed, stabilizer treatment, antiseptic are processed method simultaneously or the eukaryotic of the recombinant expressed influenza neuraminidase of selectivity after processing.
6. the method for quick of influenza neuraminidase antibody according to claim 1, is characterized in that described sample behaviour to be checked or animal body fluid.
7. the method for quick of influenza neuraminidase antibody according to claim 6, is characterized in that serum, bronchial perfusate, blood plasma or tissue fluid that described sample to be checked is human or animal.
8. the method for quick of influenza neuraminidase antibody according to claim 1, is characterized in that described influenza neuraminidase antibody is one or more the potpourri in IgG, IgM, IgA, IgE, IgD.
9. according to the method for quick of the influenza neuraminidase antibody described in claim 1 ~ 8 any one, it is characterized in that described recombinant expressed be transient expression or stably express.
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| GB0406013D0 (en) * | 2004-03-17 | 2004-04-21 | Chiron Srl | Analysis of saccharide vaccines without interference |
| CN103450350B (en) * | 2013-07-30 | 2015-07-01 | 中国人民解放军军事医学科学院基础医学研究所 | Epitope antigens of human infection with H7N9 avian influenza and applications of epitope antigens in immune detection reagent |
| CN104312983B (en) * | 2014-10-20 | 2017-04-12 | 中国疾病预防控制中心病毒病预防控制所 | Recombinant influenza virus and application thereof |
| JP6772157B2 (en) | 2015-02-05 | 2020-10-21 | ヤンセン ファッシンズ アンド プリベンション ベーフェーＪａｎｓｓｅｎ Ｖａｃｃｉｎｅｓ ＆ Ｐｒｅｖｅｎｔｉｏｎ Ｂ．Ｖ． | Binding molecule to influenza hemagglutinin and its use |
| US10174127B2 (en) | 2015-02-05 | 2019-01-08 | Janssen Vaccines & Prevention B.V. | Human neutralizing antibodies binding to influenza neuraminidase |
| CN110095593A (en) * | 2019-04-28 | 2019-08-06 | 深圳贝安基因生物科技有限公司 | A kind of double antibodies sandwich-enzyme-linked immunosorbent assay kit detecting H5N8 subtype avian influenza virus neuraminidase |
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