CN102645539B - HA (hemagglutinin) epitope-ELISA (enzyme-linked immunosorbent assay) kit for H5-subtype AIV (avian influenza virus) antibodies - Google Patents
HA (hemagglutinin) epitope-ELISA (enzyme-linked immunosorbent assay) kit for H5-subtype AIV (avian influenza virus) antibodies Download PDFInfo
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Abstract
The invention discloses an HA (hemagglutinin) epitope-ELISA (enzyme-linked immunosorbent assay) kit for H5-subtype AIV (avian influenza virus) antibodies. The kit can be used for detecting or assisting in detecting antisera of H5-subtype avian influenza viruses, and the kit internally contains an independently-packaged protein shown in the following a) or b) as a detecting antigen: a) a protein consisting of amino acid sequences shown as a sequence 2 in a sequence table; and b) a protein which is formed by that amino acid sequences (shown as the sequence 2 in the sequence table) suffer from substitution and/or deletion and/or addition through one or more amino acid residues and can carry out specific binding with antisera of H5-subtype avian influenza viruses. Experiments show that the kit disclosed by the invention can specifically detect to-be-detected animal antisera containing H5-subtype avian influenza viruses, the sensitivity reaches 1:800, and the accuracy rate reaches more than 92%; the kit is suitable to be used for detecting antisera of H5-subtype avian influenza viruses from chickens, ducks, gooses, wild birds, pigs, cattle and persons and the like; the kit both can be applied to the early diagnosis of H5-subtype avian influenzas, and can be applied to antibody monitoring; and the kit is extremely practical in the aspects of epidemiological survey and antibody monitoring in the actual production.
Description
Technical field
The present invention relates to a kind of HA epitope-ELISA kit of H5 subtype avian influenza virus antibody.
Background technology
Avian influenza virus (Avian influenza virus, AIV) is a kind of A type influenza virus that can cause bird generation serious acute infectious disease, and its bird flu causing is classified as class A zoonosis by International Office of Epizootics (OIE).This virus is easy to morph, and has found so far 16 hemagglutinin (HA) and 9 neuraminidases (NA) hypotype.Wherein, H5 is a kind of highly pathogenic hypotype AIV, can cause the extensive death of bird and cause huge economic loss to aviculture.Evidence suggests, H5 hypotype AIV can also propagate to the mankind and other multiple mammal by number of ways, as: cat, dog, pig and tiger etc., cause its morbidity even dead.Therefore, quick and precisely detecting H5 subtype avian influenza virus, to infect investigation and prevention and control to this disease significant.
AIV genome by 8 independently RNA form, the 10 kinds of albumen of encoding altogether.Wherein, HA albumen is the important surface antigen albumen of of AIV, in viral absorption with wear film, determine that the aspects such as viral pathogenicity play an important role, be also that virus stimulates body to produce the important antigen of neutralizing antibody, significant in the immunology diagnosis of AIV.
Up to now, about this sick serodiagnosis comprises hemagglutination-inhibition test (HI), agar gel diffusion test (AGP), immunofluorescence technique (IFA), enzyme linked immunosorbent assay (ELISA) etc.
Hemagglutination-inhibition test (HI) is the common method that bird flu and hypotype thereof are identified, but complicated operation is time-consuming, need to carry out with hemagglutination test (HA) simultaneously, and easily there is cross reaction for the virus infections with identical neuraminidase hypotype (NA), cause mistaken diagnosis.
Agar gel diffusion test (AGP) is to utilize soluble antigen antibody to spread in Ago-Gel, when antigen-antibody generation immune response, forms macroscopic immune complex precipitation line in Ago-Gel, and result of determination according to this.The method poor specificity, susceptibility are low, easily cause mistaken diagnosis.
Immunofluorescence technique (IFA) is a kind of immunological method and fluorescence labeling technology to be combined to the method that research differential protein antigen distributes in cell.The method as far back as 1961 for the para-influenzal quick diagnosis of people, in the diagnosis of bird flu, also have application.The method has the features such as sensitivity, quick, easy, cost is low, but also exists non-specific fluorescence to disturb and the false positive results problem of appearance.
Enzyme linked immunosorbent assay (ELISA) is a class take enzyme-labelled antigen or enzyme labelled antibody as main agents, by the substrate for enzymatic activity colour generation in compound, checking matter is carried out to qualitative or quantitative immuno-labelling technique.The features such as that this technology has is highly sensitive, high specificity, accuracy are good, simple to operation are the common technologies to viral antigen and antibody test thereof at present.This technology is early than the detection that is applied to bird flu for 1974 by European countries, and the bird flu ELISA diagnostic method of China's research and development at present comprises that avian flu virus detection ELISA and avian influenza antibody detect two kinds of ELISA.The antigen that these two kinds of technology are used is totivirus concentrated antigen, need to prepare by techniques such as concentrated, purifying and deactivations.Avian influenza virus is a kind of virus that is easy to variation, has the harm of potentiality bio-safety.Especially H5 subtype avian influenza virus, is a kind of highly pathogenic virus, requires to produce in the bio-safety facility of P3 level at least while preparing antigen.Production cost is large, instrument and equipment requirement is high, can not meet needs of production, is unfavorable for applying of these technology.For these problems, there is scholar to set up bird flu ELISA detection technique take hemagglutinin (HA) gene expression albumen as antigen, and proved that the HA hypotype can be used for avian influenza virus carries out somatotype.But owing to containing more Escherichia coli rare codon in HA gene, when HA full-length gene is expressed in prokaryotic, in sequence, the rare codon of dense distribution can have a significant impact the expression output of recombinant protein, cannot meet need of production; And these rare codons are widely distributed, it is transformed and need to expend very large man power and material, increase considerably R&D costs.
Summary of the invention
An object of the present invention is to provide HA epitope-ELISA kit that a kind of H5 subtype avian influenza virus infects serum antibody.This kit can be used for detecting or the antiserum of auxiliary detection tested animal H5 subtype avian influenza virus, and following albumen a) or b) that contains independent packaging in described kit is as detectable antigens:
A) protein being formed by amino acid sequence shown in sequence table sequence 2;
B) by the amino acid sequence of sequence table sequence 2 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and can with the albumen of the antiserum specific bond of H5 subtype avian influenza virus.
Amino acid sequence shown in sequence table sequence 2 is comprised of 244 amino acid residues, the HA epitope that contains H5 subtype avian influenza virus.
Albumen in above-mentioned in order to make (a) is convenient to purifying, can connect label as shown in table 1 at the amino terminal of the protein being comprised of the amino acid sequence shown in sequence table sequence 2 or carboxyl terminal.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Described albumen specifically can be the albumen that adds 6 histidines at the amino terminal of amino acid sequence shown in sequence table sequence 2.
In described kit, also contain the coating buffer of independent packaging and/or the PBST damping fluid of independent packaging and/or the TMB storage liquid of the confining liquid of independent packaging and/or the sample diluting liquid of independent packaging and/or independent packaging and/or the tmb substrate dilution of independent packaging and/or the stop buffer of independent packaging;
The solvent of described coating buffer is water, and solute is Na
2cO
3and NaHCO
3, solute Na
2cO
3and NaHCO
3concentration in described coating buffer is respectively 1.59g/L and 2.93g/L, and described coating buffer pH value specifically can be 9.6;
The solvent of described PBST damping fluid is water, and solute is NaCl, KCl, KH
2pO
4, Na
2hPO
4and Tween-20, solute NaCl, KCl, KH
2pO
4and Na
2hPO
4concentration in described PBST damping fluid is respectively 8.0g/L, 0.2g/L, 0.2g/L and 1.15g/L, and the volumn concentration of described Tween-20 in described PBST damping fluid is 0.05%, and the pH value of described PBST damping fluid specifically can be 7.4;
The solvent of described confining liquid is PBST damping fluid, and solute is skim milk, and the concentration of solute skim milk in described confining liquid is 50g/L;
The solvent of described sample diluting liquid is PBST damping fluid, and solute is bovine serum albumin(BSA), and the concentration of solute bovine serum albumin(BSA) in described sample diluting liquid is 10g/L;
The solvent of described TMB storage liquid is absolute ethyl alcohol, and solute is tetramethyl benzidine, and the concentration of solute tetramethyl benzidine in described TMB storage liquid is 2mg/mL;
The solvent of described tmb substrate dilution is water, and solute is Na
2hPO
4and citric acid, solute Na
2hPO
4be respectively 18.27g/L and 4.665g/L with the concentration of citric acid in described tmb substrate dilution, the pH value of described tmb substrate dilution specifically can be 5.5;
Described stop buffer is the aqueous sulfuric acid of 2mol/L.
In described kit, also contain the antibody of the anti-tested animal IgG of the enzyme labeling of independent packaging.
The marker enzyme of the antibody of the anti-tested animal IgG of described enzyme labeling is horseradish peroxidase.
Described tested animal specifically can be chicken or duck, also can be goose, pig, ox, people and wild bird.
Detectable antigens in enzyme linked immunological kit provided by the present invention is the HA epitope RT-PCR expressing protein of H5N1 subtype avian influenza virus, this recombinant expression protein is not change under the prerequisite of original gene information, by selecting epitope in HA gene, concentrate, express as genes of interest in the region that between rare codon, distance is relatively far away, solved HA full-length gene causes destination protein to express the difficult problem yielding poorly because subregion exists the rare codon of dense distribution in expression process, opened up a convenience, cheap, the approach of effective acquisition detectable antigens.
Experimental results show that, kit of the present invention can detect the tested animal antiserum that contains H5 subtype avian influenza virus specifically, sensitivity reaches 1: 800, rate of accuracy reached is more than 92%, this kit is applicable to detect the H5 subtype avian influenza virus antiserum in the sources such as chicken, duck, goose, wild bird, pig, ox and people, both can be used for the early diagnosis of H5 subtype avian influenza, can be used for again antibody detection, very practical aspect the epidemiology survey in production reality and antibody detection.
Accompanying drawing explanation
Fig. 1 is the amino acid sequence of the H5N1 HA Gene of H 9 Subtype AIV coding codon service condition table (critical value is 1%) in Escherichia coli.Wherein, the full length sequence shown in figure is the amino acid sequence of H5N1 HA Gene of H 9 Subtype AIV open reading frame coding, and dash area is the amino acid sequence of HA antigen epitope genes coding of the present invention; In sequence, the Escherichia coli rare codon of frequency of utilization≤1% shows with capitalization; The Escherichia coli rare codon of frequency of utilization≤0.28% shows with the capitalization with frame.
Fig. 2 is the pcr amplification product electrophoretogram of open reading frame (ORF) gene of H5 subtype avian influenza virus HA antigen epitope genes fragment and HA gene.Wherein, the HA antigen epitope genes fragment that swimming lane 1 contains EcoR I restriction endonuclease recognition sequence for two ends, swimming lane 2 is molecular weight standard, be followed successively by from top to bottom 1500bp, 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp, the HA gene open reading frame fragment that swimming lane 3 contains EcoR I restriction endonuclease recognition sequence for two ends.
Fig. 3 is the SDS-PAGE electrophoretogram of H5 subtype avian influenza virus HA epitope recombinant expression protein.Wherein, swimming lane 1 is pre-dsred protein MarkerIII, is followed successively by from top to bottom 94KD, 60KD, 45KD, 27KD and 18KD; Swimming lane 2 is not induced contrast for recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA, swimming lane 3 is the inclusion body of recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA, and arrow indication place is destination protein.
Fig. 4 is the SDS-PAGE electrophoretogram of H5 HA Gene of H 9 Subtype AIV open reading frame recombinant expression protein.Wherein, swimming lane 1 is pre-dsred protein MarkerIII, is followed successively by from top to bottom 94KD, 60KD, 45KD and 27KD; Swimming lane 2 is not induced contrast for recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF, swimming lane 3 is the inclusion body of recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF, and arrow indication place is destination protein.
Fig. 5 is the Western-Blot analysis result of H5 subtype avian influenza virus HA epitope recombinant expression protein.Wherein, swimming lane 1 is pre-dsred protein MarkerIII, is followed successively by from top to bottom 94KD, 60KD, 45KD, 27KD and 18KD; Swimming lane 2 is the reaction result of HA epitope recombinant expression protein and H5 subtype avian influenza virus positive serum; Swimming lane 3 is the negative chicken serum reaction result of HA epitope recombinant expression protein and SPF.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The source of following embodiment serum used is as follows: H5 subtype avian influenza chicken positive serum: purchased from Harbin Wei Ke biotechnology development company, through hemagglutination-inhibition test method, identify and be positive.
H7 subtype avian influenza chicken positive serum: purchased from Harbin Wei Ke biotechnology development company, identify and be positive through hemagglutination-inhibition test method.
H9 subtype avian influenza chicken positive serum: purchased from Harbin Wei Ke biotechnology development company, identify and be positive through hemagglutination-inhibition test method.
The positive chicken serum of infective rhinitis: purchased from China Veterinery Drug Inspection Office, identify and be positive through agar gel diffusion test method.
The positive chicken serum of ewcastle disease: herd development in science and technology center purchased from Beijing Kang Nongxing, identify and be positive through hemagglutination-inhibition test method.
The positive chicken serum of egg drop syndrome: herd development in science and technology center purchased from Beijing Kang Nongxing, identify and be positive through agar gel diffusion test method.
The positive chicken serum of infective bronchitis: purchased from suitable suddenly biotechnology (Beijing) company limited, identify and be positive through agar gel diffusion test method.
HA epitope-ELISA kit for detection of H5 subtype avian influenza virus infection serum antibody of the present invention is comprised of HA epitope recombinant protein (abbreviation envelope antigen), coating buffer, PBST damping fluid, confining liquid, sample diluting liquid, TMB storage liquid, tmb substrate dilution and the stop buffer of H5 subtype avian influenza virus, its preparation method or composed as follows:
One, preparation and the evaluation of the HA epitope recombinant protein of envelope antigen-H5 subtype avian influenza virus
1, the clone of H5 HA Gene of H 9 Subtype AIV
The duck hepatic tissue that is accredited as H5N1 avian influenza virus A/Duck/GX/1/00 (H5N1) infection take normal experiment methods such as the separation of process virus, hemagglutination-inhibition tests is as the total RNA of sample extraction, reverse transcription obtains cDNA, take 5 '-atggagaaaatagtgcttc-3 ' and 5 '-ttaaatgcaaattctgcactgt-3 ' as primer, carry out again pcr amplification, obtain the DNA fragmentation that the size shown in sequence table sequence 1 is 1704bp, this sequence is the open reading frame (ORF) of H5N1 HA Gene of H 9 Subtype AIV.
2, the rare codon analysis of H5 HA Gene of H 9 Subtype AIV
Analyze open reading frame (its sequence is as shown in sequence table sequence 1) and the rare codon occurrence number of HA antigen epitope genes (its sequence is as the 289th of sequence table sequence 1 the to as shown in the of the 1020th) and the distribution situation in gene order of H5N1 HA Gene of H 9 Subtype AIV.Result is as shown in Fig. 1 and table 2.
Table 2.HA gene is in the rare codon analysis of expression in escherichia coli
Note: e. coli codon frequency of utilization is lower, and foreign gene is more beyond expression of words.
Result shows, in 567 amino acid code of the amino acid sequence total length of the open reading frame coding of H5N1 HA Gene of H 9 Subtype AIV, frequency of utilization≤1% have 156, frequency of utilization≤0.28% have 23, part rare codon close proximity, even adjacent appearance arranged side by side.Especially 3 frequencies of utilization that occur side by side the 341st the-the 343rd position (Fig. 1) are only 0.28% rare codon RRR; In 244 amino acid code of HA antigen epitope genes coding, one of frequency of utilization≤1% has 70, frequency of utilization≤0.28% have 9, these rare codons distribute compared with HA ORF gene codon, are relatively conducive to the expression of destination protein in Escherichia coli.Especially 3 frequencies of utilization having avoided (Fig. 1) on the 341st the-the 343rd position are only 0.28% rare codon RRR, effectively reduce the probability of protein translation premature termination.
3, the acquisition of H5 subtype avian influenza virus HA epitope recombinant protein
1) construction of recombinant expression plasmid
At DNA fragmentation two ends shown in the 289th to the 1020th of sequence table sequence 1, add respectively the sequence " ctgatatcggatccgtc " (this sequence is the sequence of EcoR I restriction enzyme site one side of plasmid pET-32a (+)) of 17bp and the sequence " tgtcgacggagctcg " (this sequence is the sequence of the EcoR I restriction enzyme site opposite side of plasmid pET-32a (+)) of 17bp, obtain the DNA fragmentation of 766bp, electrophoresis result is as shown in the swimming lane 1 of Fig. 2; With EcoR I restriction enzyme, by after plasmid pET-32a (+) (Novagen company) linearization, then the DNA fragmentation of this 766bp and linearizing plasmid pET-32a (+) (Novagen company) are utilized
recombinant clone kit (L00339, Nanjing Genscript Biotechnology Co., Ltd.) connect, transform again Rosetta-gami B (DE3) (Novagen company), extract positive colony plasmid, through sequence verification, obtained by the recombinant expression plasmid of DNA fragmentation shown in the 289th to the 1020th of the EcoR I site catenation sequence table sequence 1 at plasmid pET-32a (+) multiple clone site place, by this recombinant expression plasmid called after pET-32a (+)-HA.The albumen (being the i.e. albumen of 97-340 amino acids composition of dash area in Fig. 1) of 244 amino acid compositions shown in the 289th to the 1020th of sequence table sequence 1 shown in DNA fragmentation code sequence list sequence 2.
At the DNA fragmentation two ends of 1704bp shown in sequence table sequence 1, add respectively the sequence " ctgatatcggatccgtc " of 17bp and the sequence " tgtcgacggagctcg " of 17bp, obtain the DNA fragmentation electrophoresis result of 1738bp as shown in the swimming lane 3 of Fig. 2; With EcoR I restriction enzyme, by after plasmid pET-32a (+) (Novagen company) linearization, then the DNA fragmentation of this 1738bp and linearizing plasmid pET-32a (+) (Novagen company) are utilized
recombinant clone kit (L00339, Nanjing Genscript Biotechnology Co., Ltd.) connect, transform again Rosetta-gami B (DE3) (Novagen company), extract positive colony plasmid, through sequence verification, obtained by the recombinant expression plasmid of DNA fragmentation shown in the EcoR I site catenation sequence table sequence 1 at plasmid pET-32a (+) multiple clone site place, by this recombinant expression plasmid called after pET-32a (+)-HAORF.The albumen being formed by 567 amino acid shown in DNA fragmentation code pattern 1 shown in sequence table sequence 1.
2) abduction delivering of recombinant protein
PET-32a (+)-HA is proceeded in bacterial strain Rosetta-gami B (DE3) (Novagen company), obtain recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA that contains recombinant expression plasmid pET-32a (+)-HA.
PET-32a (+)-HAORF is proceeded in bacterial strain Rosetta-gami B (DE3) (Novagen company), obtain recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF that contains recombinant expression plasmid pET-32a (+)-HAORF.
This recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA and Rosetta-gami B (DE3) are cultivated respectively as follows, collect bacterial sediment:
Be inoculated into the LB fluid nutrient medium of 5mL containing 50 μ g/mL ampicillins, 37 ℃ of overnight incubation, by 10% inoculum concentration, be inoculated into 500mL containing in the LB fluid nutrient medium of 50 μ g/mL ampicillins again, 37 ℃, 150 revs/min of joltings are cultured to exponential phase, with ultraviolet spectrophotometer, measure the OD600 light absorption value of bacterium liquid, when light absorption value reaches any one value between 0.5～0.8, adding final concentration is isopropyl-β-D-sulfo-galactopyranoside (IPTG) derivant of 1mmol/L, continuation is at 37 ℃, 150 revs/min of jolting overnight incubation, obtain bacteria suspension, the centrifugal 10min of 2500 × g, collect bacterial sediment.
3) purifying of recombinant protein
By step 2) recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA obtaining and the bacterial sediment of Rosetta-gami B (DE3)/pET-32a (+)-HAORF be handled as follows respectively: with the PBS solution of 10mL, (solvent is water, the Na of the NaCl that solute is 8g/L, the KCl of 0.2g/L, 1.44g/L
2hPO
4kH with 0.24g/L
2pO
4, with HCl, adjusting pH is 7.4) and resuspended, add final concentration is the lysozyme (the green skies, Shanghai Bioisystech Co., Ltd) of 100 μ g/mL simultaneously, carries out ultrasonic treatment on ice, the centrifugal collection inclusion body precipitation of 10000 × g.
Get the inclusion body precipitation of recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA and carry out SDS-PAGE evaluation, take recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA of not carrying out IPTG abduction delivering as contrast, result as shown in Figure 3; Get the inclusion body precipitation of recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF and carry out SDS-PAGE evaluation, take recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF of not carrying out IPTG abduction delivering as contrast, result as shown in Figure 4.
In Fig. 3,6 histidine-tagged proteins of the expressed destination protein of recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA and pET-32a (+) carrier form the fusions (called after HA epitope recombinant protein) that molecular weight is 48.1KD, further gel image scanning analysis shows, the destination protein of this 48.1KD accounts for 35% of bacterial protein; In Fig. 4, to form molecular weight be 84.7KD (called after HA recombinant protein) to 6 histidine-tagged proteins of the expressed destination protein of recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HAORF and pET-32a (+) carrier, further gel image scanning analysis shows, the destination protein of this 84.7KD accounts for 8% of bacterial protein, illustrates that the prokaryotic expression amount of HA epitope recombinant protein that HA antigen epitope genes is carried out to recombinant expressed acquisition is apparently higher than the HA recombinant protein that HA full length gene is carried out to recombinant expressed acquisition.
Utilize QIAexpress Ni-NTA Fast Sart (German QIAGEN company, Cat.306000) the 48.1KD albumen of 6 histidine marks in recombinant bacterium Rosetta-gami B (DE3)/pET-32a (+)-HA inclusion body precipitation is carried out to purifying, obtain HA epitope recombinant protein.
4) activity of recombinant protein detects
According to a conventional method, lauryl sodium sulfate-the polyacrylamide gel (SDS-PAGE) of preparation 12%, get 10 μ L steps 3) the HA epitope recombinant protein of purifying carries out electrophoresis on SDS-PAGE, then carries out Western-Blot analysis, and result is as shown in Figure 5.Result shows, HA epitope recombinant protein can hybridization colour developing occur with H5 subtype avian influenza virus positive serum, occurs the hybridization reaction band of 48.1kD, and with the negative chicken serum of SPF without any specific hybrid reaction zone.
Two, the preparation of other reagent of kit
Coating buffer: solvent is water, solute is Na
2cO
3and NaHCO
3, solute Na
2cO
3and NaHCO
3concentration in coating buffer is respectively 1.59g/L and 2.93g/L, and pH value is 9.6.
PBST damping fluid: solvent is water, solute is NaCl, KCl, KH
2pO
4, Na
2hPO
4and Tween-20, solute NaCl, KCl, KH
2pO
4and Na
2hPO
4concentration in PBST damping fluid is respectively 8.0g/L, 0.2g/L, 0.2g/L and 1.15g/L, and the volumn concentration of Tween-20 in PBST damping fluid is 0.05%, and pH value is 7.4.
Confining liquid: solvent is PBST damping fluid, solute is skim milk, the concentration of solute skim milk in confining liquid is 50g/L.
Sample diluting liquid: solvent is PBST damping fluid, solute is bovine serum albumin(BSA) (BSA), the concentration of solute BSA in sample diluting liquid is 10g/L.
TMB storage liquid: solvent is absolute ethyl alcohol, solute is tetramethyl benzidine (TMB), and the concentration of solute TMB in TMB storage liquid is 2mg/mL, and 4 ℃ keep in Dark Place.
Tmb substrate dilution: solvent is water, solute is Na
2hPO
4and citric acid, solute Na
2hPO
4be respectively 18.27g/L and 4.665g/L with the concentration of citric acid in tmb substrate dilution, pH value is 5.5.
Substrate nitrite ion: containing TMB storage liquid 0.5mL, tmb substrate dilution 10mL, H
2o
232 μ L.
Stop buffer: the aqueous sulfuric acid that concentration is 2M.
The specific detection that embodiment 2, H5 subtype avian influenza virus infect HA epitope-ELISA kit of serum antibody
The kit that uses embodiment 1 to prepare, adopt indirect ELISA method (indirect ELISA) to the hemagglutination-inhibition test of following process, the chicken serum (serum to be checked) that the normal experiment methods such as agar gel diffusion test are identified carries out the detection of H5 subtype avian influenza virus: H5 subtype avian influenza chicken positive serum (positive control), SPF chicken serum (negative control), H7 subtype avian influenza chicken positive serum (serum 1 to be checked), H9 subtype avian influenza chicken positive serum (serum 2 to be checked), the positive chicken serum of infective rhinitis (serum 3 to be checked), the positive chicken serum of ewcastle disease (serum 4 to be checked), the positive chicken serum of egg drop syndrome (serum 5 to be checked), the positive chicken serum of infective bronchitis (serum 6 to be checked), concrete steps are as follows:
1, the dilution of antigen
With BCA determination of protein concentration kit (the green skies, Shanghai Bioisystech Co., Ltd, P0012) measure in embodiment 1 step 3 3) concentration of the HA epitope recombinant protein of purifying, purifying protein is diluted to 15 μ g/ μ L with coating buffer, as envelope antigen.
2, antigen is coated
15 μ g/ μ L envelope antigens are joined to elisa plate by the amount in 100 μ L/ holes.After 37 ℃ of effect 1h, then 4 ℃ of effect 2h, PBST damping fluid is washed plate 3 times, dries for the last time.The confining liquid sealing 40min that adds 100 μ L/ holes, PBST damping fluid is washed plate 3 times, dries standby.
3, serum to be checked adds
With sample diluting liquid, by 100 times of serum dilutions to be checked, by the amount in 100 μ L/ holes, add elisa plate, 3 repetitions of each sample, 37 ℃ of effect 40min, PBST damping fluid is washed plate 3 times.
4, ELIAS secondary antibody adds
With sample diluting liquid by anti-the goat-anti chicken IgG of horseradish peroxidase-labeled bis-(KPL company, 14-24-06) dilution adds each reacting hole by the amount in 100 μ L/ holes after 5000 times, 37 ℃ of effect 40min, PBST damping fluid is washed plate 3 times, distillation washing plate 1 time.
5, colour developing
By the amount in 100 μ L/ holes, to each reacting hole, add substrate nitrite ion, 37 ℃ of effect 5min, add 50 μ l stop buffers.
6, result detects
ELISA Plate is placed in to microplate reader, measures the OD value of 450nm.According to formula X+3STD, calculate positive decision content, the negative control sample OD of X in formula
450mean value, the negative control sample variance of STD.As the OD of serum to be checked
450during mean value>=positive decision content, judge that serum to be checked is as H5 subtype avian influenza virus positive serum, result is as shown in table 3.
Indirect ELISA testing result (the OD of table 3. kit of the present invention
450)
According to upper table, calculate and learn that X is that 0.0750, STD is 0.006, positive decision content X+3STD is 0.093.
The result of table 3 shows, the specificity of kit HA epitope recombinant protein of the present invention is fine, the antiserum generation specific binding reaction that can infect with H5 subtype avian influenza virus.
The sensitivity that embodiment 3, H5 subtype avian influenza virus infect HA epitope-ELISA kit of serum antibody detects
One, the sensitivity of envelope antigen in kit of the present invention
Envelope antigen in kit of the present invention is diluted to respectively to 20 μ g/ μ L, 15 μ g/ μ L, 10 μ g/ μ L, 5 μ g/ μ L, 2 μ g/ μ L, 1 μ g/ μ L, 0.5 μ g/ μ L, 0.25 μ g/ μ L according to the method for embodiment 2 steps 1, the coated elisa plate of antigen coated method of pressing step 2 in embodiment 2, each dilutability repeats 3 times.H5 subtype avian influenza chicken positive serum (positive control) and the SPF chicken serum (negative control) through hemagglutination-inhibition test method, identified are diluted respectively to 100 times with sample diluting liquid, by the sensitivity of embodiment 2 step 3-6 time-and-motion study envelope antigens.Result is as shown in table 4, and in table, numerical value is the mean value repeating for 3 times.
Sensitivity testing result (the OD of table 4.H5 subtype avian influenza virus HA epitope recombinant protein
450)
Note: sample OD
450positive when value is greater than positive decision content, negative while being less than positive decision content.
Table 4 result shows, the H5 subtype avian influenza virus HA epitope recombinant protein of 0.25 μ g/ μ L can detect the positive serum of 100 times of dilutions.
Two, kit of the present invention detects the sero-fast sensitivity of H5 subtype avian influenza virus
By identifying the positive chicken serum of H5 subtype avian influenza (positive control) through hemagglutination-inhibition test method and being accredited as negative SPF chicken serum (negative control), with sample diluting liquid, carry out 50,100,200,300,400,500,600,700,800,900 and 1000 times of dilutions respectively, HA epitope recombinant protein take concentration as 15 μ g/ μ L is as envelope antigen, according to the method for embodiment 2, above-mentioned diluted feminine gender and positive are detected respectively, the sample of each concentration repeats 3 times, and result is as shown in table 5.
Table 5. kit of the present invention detects the sero-fast sensitivity result of H5 subtype avian influenza virus (OD
450)
The result of table 5 shows, kit of the present invention reaches 1: 800 when the indirect ELISA detection sensitivity of the chicken antiserum that the H5 subtype avian influenza virus HA epitope recombinant protein using 15 μ g/ μ L infects H5 subtype avian influenza virus as envelope antigen.
The accuracy rate that embodiment 4, H5 subtype avian influenza virus infect HA epitope-ELISA kit of serum antibody detects
One, kit monitoring chicken group's of the present invention accuracy rate
Use kit of the present invention method in accordance with the following steps to monitor chicken group, with the negative contrast of SPF chicken serum, with the positive contrast of H5 subtype avian influenza chicken positive serum, each sample repeats for 3 times, and result represents with mean value:
1, the collection of blood serum sample to be checked
According to a conventional method, with the syringe of 2mL specification, at random from chicken group's to be checked chicken wings radicular vein blood sampling 1mL, separation of serum, saves backup in 4 ℃.
2, the dilution of antigen
Method is with the step 1 of embodiment 2.
3, antigen is coated
Method is with the step 2 of embodiment 2.
4, serum to be checked adds
Method is with the step 3 of embodiment 2.
5, ELIAS secondary antibody adds
With sample diluting liquid by anti-the goat-anti chicken IgG of horseradish peroxidase-labeled bis-(KPL company, 14-24-06) dilution adds each reacting hole by the amount in 100 μ L/ holes after 5000 times, 37 ℃ of effect 40min, PBST damping fluid is washed plate 3 times, distillation washing plate 1 time.
6, colour developing
Method is with the step 5 of embodiment 2.
7, result judgement
Method is with the step 6 of embodiment 2.
Result: in 52 parts of chicken serum samples, the sample that is the H5 subtype avian influenza virus positive has 47 parts, and the sample that is H5 subtype avian influenza virus feminine gender has 5 parts, and testing result is as shown in table 6 and table 7; Bird flu H5 antibody assay kit (the Avian Influenza H5 Antibody Test Kit that is simultaneously antigen in order to H5 subtype avian influenza virus by above-mentioned 52 parts of chicken serum samples, 06-40979-02, Idex ASA) detect, the sample that result is the H5 subtype avian influenza virus positive has 49 parts, and the sample that is H5 subtype avian influenza virus feminine gender has 3 parts.
Result shows: kit of the present invention is compared with bird flu H5 antibody assay kit take H5 subtype avian influenza virus as antigen, and the coincidence rate that detects the H5 subtype avian influenza virus antibody in chicken serum is 96.15%.
ELISA testing result (the OD of table 6. kit of the present invention to chicken group
450)
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| A | 0.095 | 0.120 | 0.179 | 0.142 | 0.217 | 0.127 | 0.165 |
| B | 0.098 | 0.122 | 0.342 | 0.213 | 0.188 | 0.171 | 0.274 |
| C | 0.221 | 0.177 | 0.257 | 0.282 | 0.301 | 0.097 | 0.189 |
| D | 0.246 | 0.277 | 0.126 | 0.159 | 0.199 | 0.143 | 0.150 |
| E | 0.149 | 0.197 | 0.124 | 0.257 | 0.176 | 0.122 | 0.239 |
| F | 0.164 | 0.112 | 0.200 | 0.219 | 0.083 | 0.274 | 0.304 |
| G | 0.219 | 0.122 | 0.092 | 0.300 | 0.252 | 0.079 | 0.111 |
| H | 0.237 | 0.119 | 0.108 | 0.158 | 0.130 | 0.112 | 0.097 |
The negative results of comparison of note: A1 and B1, the positive results of comparison of C1 and D1, all the other are chicken serum sample result to be measured; According to negative control result in table, calculate and learn that X is that 0.0965, STD is 0.002121, positive decision content X+3STD is 0.1014, sample OD
450positive when value is greater than positive decision content 0.1014, negative while being less than positive decision content 0.1014.
The ELISA testing result (the moon/sun) of table 7. kit of the present invention to chicken group
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| A | Negative control | Positive | Positive | Positive | Positive | Positive | Positive |
| B | Negative control | Positive | Positive | Positive | Positive | Positive | Positive |
| C | Positive control | Positive | Positive | Positive | Positive | Negative | Positive |
| D | Positive control | Positive | Positive | Positive | Positive | Positive | Positive |
| E | Positive | Positive | Positive | Positive | Positive | Positive | Positive |
| F | Positive | Positive | Positive | Positive | Negative | Positive | Positive |
| G | Positive | Positive | Negative | Positive | Positive | Negative | Positive |
| H | Positive | Positive | Positive | Positive | Positive | Positive | Negative |
Two, kit monitoring duck group's of the present invention accuracy rate
Use kit of the present invention method in accordance with the following steps to monitor duck group, negative control is nonimmune healthy duck serum, through hemagglutination-inhibition test, prove H5 subtype avian influenza feminine gender, positive control is H5N1 hypotype recombinant fowl influenza virus inactivated vaccine (H5N1Re_5 strain) (YEBIO Bioengineering Co., Ltd of Qingdao) immune duck serum, through hemagglutination-inhibition test, identify and be positive, each sample repeats for 3 times, and result represents with mean value:
1, the collection of blood serum sample to be checked
According to a conventional method, use the syringe of 2mL specification from duck group's to be measured duck wing radicular vein blood sampling 1mL, separation of serum, saves backup in 4 ℃.
2, the dilution of antigen
Method is with the step 1 of embodiment 2.
3, antigen is coated
Method is with the step 2 of embodiment 2.
4, serum to be checked adds
Method is with the step 3 of embodiment 2.
5, ELIAS secondary antibody adds
With sample diluting liquid by anti-anti-the rabbit of horseradish peroxidase-labeled duck IgG bis-(KPL company, 04-25-06) dilution adds each reacting hole by the amount in 100 μ L/ holes after 1000 times, 37 ℃ of effect 40min, PBST damping fluid is washed plate 3 times, distillation washing plate 1 time.
6, colour developing
Method is with the step 5 of embodiment 2.
7, result judgement
Method is with the step 6 of embodiment 2.
Result: in 52 parts of duck blood serum samples, be 37 parts of the H5 subtype avian influenza virus positive; Be 15 parts of H5 subtype avian influenza virus feminine gender, result is as shown in table 8 and table 9; Use avian influenza virus H5 hypotype hemagglutination-inhibition test antigen (Harbin Wei Ke biotechnology development company) to carry out hemagglutination-inhibition test above-mentioned duck blood serum sample simultaneously, result is 41 parts of the H5 subtype avian influenza virus positive, is 11 parts of H5 subtype avian influenza virus feminine gender.
The above results shows, compared with hemagglutination-inhibition test, the coincidence rate that uses kit of the present invention to detect the H5 subtype avian influenza virus antibody in duck serum is 92.30%.
Testing result (the OD of table 8. kit of the present invention to duck group
450)
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| A | 0.614 | 0.097 | 0.095 | 0.095 | 0.094 | 0.088 | 0.315 |
| B | 0.673 | 0.435 | 0.376 | 0.394 | 0.278 | 0.263 | 0.298 |
| C | 0.081 | 0.106 | 0.073 | 0.089 | 0.571 | 0.182 | 0.071 |
| D | 0.075 | 0.132 | 0.144 | 0.160 | 0.114 | 0.207 | 0.175 |
| E | 0.123 | 0.064 | 0.059 | 0.125 | 0.047 | 0.254 | 0.085 |
| F | 0.081 | 0.071 | 0.173 | 0.086 | 0.093 | 0.071 | 0.106 |
| G | 0.154 | 0.300 | 0.297 | 0.341 | 0.281 | 0.214 | 0.059 |
| H | 0.193 | 0.070 | 0.259 | 0.055 | 0.166 | 0.154 | 0.258 |
The positive results of comparison of note: A1 and B1, the negative results of comparison of C1 and D1, calculates and learns that the X of negative control is that 0.078, STD is 0.0042426 according to this table, and positive decision content X+3STD is 0.0907279; Sample OD
450positive when value is greater than positive decision content 0.0907279, negative while being less than positive decision content 0.0907279.
The testing result (the moon/sun) of table 9. kit of the present invention to duck group
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| A | Positive control | Positive | Positive | Positive | Positive | Negative | Positive |
| B | Positive control | Positive | Positive | Positive | Positive | Positive | Positive |
| C | Negative control | Positive | Negative | Negative | Positive | Positive | Negative |
| D | Negative control | Positive | Positive | Positive | Positive | Positive | Positive |
| E | Positive | Negative | Negative | Positive | Negative | Positive | Negative |
| F | Negative | Negative | Positive | Negative | Positive | Negative | Positive |
| G | Positive | Positive | Positive | Positive | Positive | Positive | Negative |
| H | Positive | Negative | Positive | Negative | Positive | Positive | Positive |
Claims (7)
1. detect or the sero-fast enzyme linked immunological kit of auxiliary detection tested animal H5 subtype avian influenza virus, it is characterized in that: the protein being formed by amino acid sequence shown in sequence table sequence 2 that contains independent packaging in described kit.
2. kit according to claim 1, is characterized in that: described protein is the albumen that adds 6 histidines at the amino terminal of amino acid sequence shown in sequence table sequence 2.
3. kit according to claim 1 and 2, is characterized in that: in described kit, also contain the coating buffer of independent packaging and/or the PBST damping fluid of independent packaging and/or the TMB storage liquid of the confining liquid of independent packaging and/or the sample diluting liquid of independent packaging and/or independent packaging and/or the tmb substrate dilution of independent packaging and/or the stop buffer of independent packaging;
The solvent of described coating buffer is water, and solute is Na
2cO
3and NaHCO
3, solute Na
2cO
3and NaHCO
3concentration in described coating buffer is respectively 1.59g/L and 2.93g/L;
The solvent of described PBST damping fluid is water, and solute is NaCl, KCl, KH
2pO
4, Na
2hPO
4and Tween-20, solute NaCl, KCl, KH
2pO
4and Na
2hPO
4concentration in described PBST damping fluid is respectively 8.0g/L, 0.2g/L, 0.2g/L and 1.15g/L, and the volumn concentration of described Tween-20 in described PBST damping fluid is 0.05%;
The solvent of described confining liquid is PBST damping fluid, and solute is skim milk, and the concentration of solute skim milk in described confining liquid is 50g/L;
The solvent of described sample diluting liquid is PBST damping fluid, and solute is bovine serum albumin(BSA), and the concentration of solute bovine serum albumin(BSA) in described sample diluting liquid is 10g/L;
The solvent of described TMB storage liquid is absolute ethyl alcohol, and solute is tetramethyl benzidine, and the concentration of solute tetramethyl benzidine in described TMB storage liquid is 2mg/mL;
The solvent of described tmb substrate dilution is water, and solute is Na
2hPO
4and citric acid, solute Na
2hPO
4be respectively 18.27g/L and 4.665g/L with the concentration of citric acid in described tmb substrate dilution;
Described stop buffer is the aqueous sulfuric acid of 2mol/L.
4. kit according to claim 3, is characterized in that: described coating buffer pH value is 9.6; The pH value of described PBST damping fluid is 7.4; The pH value of described tmb substrate dilution is 5.5.
5. kit according to claim 4, is characterized in that: the antibody that also contains the anti-tested animal IgG of the enzyme labeling of independent packaging in described kit.
6. kit according to claim 5, is characterized in that: the marker enzyme of the antibody of the anti-tested animal IgG of described enzyme labeling is horseradish peroxidase.
7. kit according to claim 1, is characterized in that: described tested animal is chicken or duck.
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