CN1712965A - Differential Diagnosis of Avian Influenza Naturally Infected Chickens by Enzyme-linked Immunosorbent Assay - Google Patents
Differential Diagnosis of Avian Influenza Naturally Infected Chickens by Enzyme-linked Immunosorbent Assay Download PDFInfo
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Abstract
A method of utilizing enzyme - linked immunosorbent assay to diagnose and identify naturally chicken herd of avian flu uses pronucleus to present NS 1 protein of H9N2 avian flu virus and uses it as antigen to coat reaction plate of enzyme mark. The method can identify and diagnose naturally infected chicken herd of avian flu and immunization chicken herd by using indirect enzyme - lined immunosorbent assay to determine level of antibody IgG in serum to be tested in reaction with antigen.
Description
Technical field
What the present invention relates to is a kind of method of biological technical field, specifically, is a kind of method of antidiastole bird flu natural infection chicken group enzyme linked immunosorbent assay.
Background technology
The popular of highly pathogenic bird flu caused large quantities of poultry death and poultry product to lose.Effectively one of method of control bird flu is vaccine inoculation at present, and an index of effective evaluation immune effect is exactly the antibody horizontal after the detection immunity.But the wild virus infection later stage also can induce antibody to produce, and this moment, the chicken group was with poison, then must take the measure of slaughtering absolutely in the production to this.Therefore effectively the antibody of the antibody of differentiation natural infection generation and immunity inoculation generation is extremely important.A large amount of NS1 albumen of can finding in nucleus in early days of avian flu virus infection cell exists, and in the late period of infecting, NS1 albumen can occur in endochylema, and it can stimulate body to produce NS1 non-structural protein antibody.But in the virion of maturation, do not find this albumen.After using vaccine immunity, the NS1 protein antibodies in the chicken body, can not occur or the NS1 protein antibodies of utmost point low content occur, and in the chicken body of wild virus infection since exist virus breeding, duplicate, can occur a large amount of NS1 albumen usually, thereby induce antibody to produce.Non-structural protein and antibody thereof can be used as a vital signs (Inoue Y of virus infections body, Suzuk R, Matsuura Y etc., detecting of the expression of hepatopathy patients hepatitis C virus NS1 nitrogen end protein and antibody, [Journal of Virology]) (Inoue Y, Suzuk R, Matsuura Y, et al.Expression of the aminoterminal half of the NS1 region of the hepatitisC virus genome and detection of antibody to the expressed protein inpatients with liver disease[J] .J Gen Virol.1992,73:2151-2154).Traditional serology experimental technique presses down experiment (HA-HI), agar diffusion experiment (AGP) and existing indirect enzyme-linked immunosorbent assay method as blood clotting and blood, can only detect the antibody horizontal after the immunity.But the wild virus infection later stage also can induce antibody to produce, and can not differentiate natural infection chicken group and immunity inoculation chicken group like this.The present invention adopts the method for indirect enzyme-linked immunosorbent assay, effectively antidiastole bird flu natural infection chicken group and immunity inoculation chicken group.
In the analysis to the prior art document, utilize bird flu NS1 albumen as envelope antigen, method by indirect enzyme-linked immunosorbent assay detects in the chicken serum and antigen reactive IgG antibody level, comes antidiastole natural infection chicken group and immunity inoculation chicken group, does not see relevant report.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of method of antidiastole bird flu natural infection chicken group enzyme linked immunosorbent assay is provided.Make it utilize bird flu NS1 protein antibodies different at natural infection chicken group and immunity inoculation chicken group content, the method by indirect enzyme-linked immunosorbent assay detects in the chicken serum comes antidiastole with antigen reactive IgG antibody level.
The present invention is achieved by the following technical solutions, in the present invention H9N2 type avian influenza virus NS1 albumen carried out prokaryotic expression, with this as antigen coated enzyme reaction plate.H9N2 type avian influenza virus is carried out the RT-PCR amplification, obtain the purpose segment, again the purpose segment is connected with pET-32a (+) carrier, obtain recombinant expression plasmid, the importing e. coli bl21 is expressed, and obtains the fusion that molecular weight is 44.2KD, this fusion, through purifying, obtain purity higher H 9N2 type avian influenza virus NS1 albumen.The albumen that obtains as antigen coated enzyme reaction plate, is measured in the serum to be checked and antigen reactive IgG antibody level, antidiastole bird flu natural infection chicken group and immunity inoculation chicken group by the method for indirect enzyme-linked immunosorbent assay.
Step of the present invention comprises:
1., the prokaryotic expression of H9N2 type avian influenza virus NS1 albumen;
2., earlier the H9N2 type that increased avian influenza virus NS1 gene is again with amplification gene;
3., cut by enzyme and be inserted among the pET-32a (+), construct prokaryotic expression plasmid, the plasmid that builds is changed in the e. coli bl21, and IPTG abduction delivering, expression product are accredited as the fusion of molecular weight 44.2KD through SDS-PAGE electrophoresis and Westernblot;
4., the foundation of the method for indirect enzyme-linked immunosorbent assay;
5., according to the method for conventional indirect enzyme-linked immunosorbent assay, sample is detected.
The method of described indirect enzyme-linked immunosorbent assay specifically comprises and determines antigen-reactive concentration, the antibody dilution multiple, and the antigen-antibody reaction time, enzyme is marked anti-IgG antibody dilution multiple.
Antigen-reactive concentration is 10 μ g/mL, and the antibody dilution multiple is 1: 200 times, and the antigen-antibody reaction time is 1h, and it is 1: 10000 that enzyme is marked anti-IgG antibody dilution multiple.
Described antigen is the antigen through escherichia coli prokaryotic expression, is coated on the mensuration of carrying out antibody on the solid phase carrier, and blood serum sample to be measured is carried out qualitative analysis.
Enzyme linked immunosorbent assay is utilized the antigen or the antibody of enzyme labeling, the antigen that carries out on solid phase carrier or the mensuration of antibody.The basis of enzyme linked immunosorbent assay is the enzyme labeling of immobilization and the antigen or the antibody of antigen or antibody.The antigen or the antibody that are combined in surface of solid phase carriers still keep its immunologic competence, and the antigen of enzyme labeling or antibody had both kept its immunologic competence, kept the activity of enzyme again.When measuring, examined sample (measuring wherein antibody or antigen) and the antigen or the antibody of surface of solid phase carriers and reacted.With the method for washing the antigen antibody complex that forms on the solid phase carrier and other materials in the liquid are separated.Add the antigen or the antibody of enzyme labeling again, also be combined on the solid phase carrier by reaction.The amount of examined object matter is certain ratio in enzyme amount on the solid phase and the sample at this moment.After adding the substrate of enzyme reaction, substrate is become coloured product by enzymatic, and the amount of product is directly related with the amount of examined object matter in the sample, so can carry out qualitative or quantitative test according to the depth of colour generation.The present invention be with avian influenza virus NS1 albumen as antigen coated enzyme reaction plate, the test serum sample is diluted and antigen-reactive according to a certain percentage, add ELIAS secondary antibody, add the substrate colour developing at last, come result of determination according to the OD value of measuring the colour developing sample.
In the present invention, term " H9N2 type avian influenza virus " is meant that influenza A virus hemagglutinin (H) and neuraminidase (N) are divided into 15 H types and 9 N types.What the present invention was used is H9N2 type avian influenza virus.
In the present invention, term " prokaryotic expression " is meant that recombinant expression plasmid imports prokaryotic, as Escherichia coli, hay bacillus etc., makes the nucleotide sequence to be expressed that inserts in the plasmid be expressed as required albumen.
In the present invention, term " fusion ", 168 amino acid that are meant Trx Tag, the His Tag, S Tag and a series of restriction enzyme sites that comprise on pET-32a (+) carrier add 0 amino acid sequence of H9N2 type bird flu NS1 protein 23, molecular weight 44.2KD.The great advantage of fusion is to make things convenient for the purifying of albumen and prevent protein degradation.
The present invention be in view of NS1 albumen in natural infection chicken body with vaccine infection chicken body in differently come antidiastole by enzyme linked immunosorbent assay.With the NS1 albumen of prokaryotic expression as antigen coated enzyme reaction plate, with serum to be checked by the antigen-reactive of certain dilution with the bag quilt.The ELIAS secondary antibody colour developing that adds anti-IgG again.Judge by the size of measuring the OD value whether the chicken group has infected bird flu.
The method that detects now bird flu mainly contains: tradition is used for the monitoring of bird flu and diagnostic mode and comprises hemagglutination test (HI), agar immunodiffusion (AGP), microneutralization, and method such as neuraminidase inhibition, check quite consuming time taking a lot of trouble.Also have some RT-PCR that combine with immunology based on molecular biology, quantitative fluorescent PCR (RRT-PCR), NASBA etc., though susceptibility is higher, complex operation, expense is higher.The diagnostic method of existing indirect enzyme-linked immunosorbent assay can not effectively be distinguished bird flu natural infection chicken group and immunity inoculation chicken group.And the method for indirect enzyme-linked immunosorbent assay provided by the present invention, combine the advantage of serological method, promptly simple to operate, cheap, having overcome the method for existing indirect enzyme-linked immunosorbent assay again can not antidiastole bird flu natural infection chicken group and immunity inoculation chicken group's shortcoming, is a kind of bird flu diagnostic method effectively reliably.
Description of drawings
Fig. 1 principle of the invention synoptic diagram
Specific embodiments
Below in conjunction with specific embodiment, the experimental technique of unreceipted actual conditions in the following fact Example, principle of work according to Fig. 1, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The prokaryotic expression of H9N2 type avian influenza virus NS1 albumen
The clone of H9N2 type bird flu NS1 gene
Trizol (Invitrogen) extracted total RNA.Carry out the RT-PCR amplification according to bird flu NS1 gene conserved sequence design primer.Be connected to pMD-18T carrier (Takara company) and go up order-checking.Comprise the primer that EcoR I restriction enzyme site and downstream comprise Xho I restriction enzyme site according to sequencing result design upstream, carry out pcr amplification.
The structure of expression vector
EcoR I and Xho I enzyme respectively cut PCR product and pET-32a (+), 10-16 ℃ of connection of spending the night of T4DNA ligase (Takara company).Connect product and change in the bacillus coli DH 5 alpha, 37 ℃ of incubated overnight are extracted plasmid.Cut evaluation with EcoR I and Xho I enzyme.The positive recombinant plasmid order-checking of identifying.
Expression of Fusion Protein
Positive colony upgrading grain changes in the e. coli bl21.1 colony inoculation 2ml of picking Amp LB nutrient culture media incubated overnight.Get 200 μ l incubated overnight bacterium liquid inoculation 20ml Amp LB nutrient culture media, 37 ℃ 150 rev/mins are shaken bacterium 2h, to OD
600Be 0.4-1.Add IPTG to final concentration be 1mM, 25 ℃ shake bacterium 4h after, collect bacterium liquid.
Detection of fusion proteins
(1) SDS-PAGE electrophoresis
The separation gel of preparation 14% and 5% concentrated glue.Sample boils 4min with sample-loading buffer boiling water.Concentrate glue voltage 80V, separation gel voltage 136V.Electrophoresis 4-5h.
Coomassie brilliant blue dyeing 2h.
The destainer decolouring.
Observe, have tangible band to occur at the 44KD place.
(2) Western Blot identifies
When the SDS-PAGE electrophoresis finishes,, wipe dried then with paper with distilled water drip washing graphite cake.
Cut 6 Whatman 3MM filter paper and a nitrocellulose filter.Nitrocellulose filter is immersed on the water surface of deionized water, filter paper is immersed in the transfering buffering liquid, balance 5min.
From the anode to the negative electrode, put correctly by the order of paper, film, glue, paper, according to 0.65mA/cm
2Energising transfer printing 5h.
Nitrocellulose filter is placed in the polybag of sealing, in add confining liquid and bird flu positive serum.(the bird flu positive serum is dilution in 1: 100).4 ℃ of night incubation change 37 ℃ again over to and hatch 2h.
Take out nitrocellulose filter PBS rinsing three times, each 10min.
Filter membrane is used TBST rinsing 10min again.
Nitrocellulose filter is placed in the polybag of sealing, in add the anti-chicken IgG two of rabbit anti-(Bethyl laboratories.Inc) of confining liquid and 1: 1000 dilution.Hatch 2h for 37 ℃.
Nitrocellulose filter is put among the TBST rinsing 3 times, each 1h.
Add substrate colour developing 2-3min.Observations has black stripe to occur at the 44KD place.
The foundation of the method for indirect enzyme-linked immunosorbent assay
Best enzyme is marked determining of anti-IgG antibody concentration
Enzyme is marked anti-IgG antibody and is done and made doubling dilution to 1 since 1: 5000: 160000, and recording ELIAS secondary antibody optimum response concentration is 1: 10000.
Best antigen-reactive concentration
Antigen is by 100 μ g/ml, 80 μ g/ml, 60 μ g/ml, 40 μ g/ml, 30 μ g/ml, 15 μ g/ml, 10 μ g/ml, 8 μ g/ml, 6 μ g/ml, 4 μ g/ml, 2 μ g/ml, 1 μ g/ml concentration dilution, antibody is done the square formation titration by dilution in 1: 50,1: 100,1: 500,1: 1000.Determine that best antigen-reactive concentration is 10 μ g/ml.
The optimum antibody extension rate
20 parts of negative serums, 11 parts of vaccine infection serum, 12 parts of natural infection serum are done times dilution in 1: 50,1: 100,1: 200,1: 500, measure OD
450, testing result is done t check, when negative serum and vaccine infection serum difference are not remarkable, and the serum diluting multiple during with natural infection serum significant difference is as best serum diluting multiple, and the optimum diluting multiple of the present invention's mensuration is 1: 200.
The antigen-antibody optimum reacting time
Antigen-antibody reacts 0.5h, 1h, 1.5h, 2h respectively, and measuring the antigen-antibody optimum reacting time is 1h.
The method antidiastole avian influenza virus natural infection chicken group of indirect enzyme-linked immunosorbent assay.
After diluted liquid was diluted to 10 μ g/ml with bag with used antigen, every hole added 100 μ l, and 4 ℃ are spent the night, 37 ℃ of 2h.Discard liquid in the hole.
Select 37 ℃ of sealings of 5% skimmed milk 2h, discard liquid in the hole, the PBST washing lotion is cleaned 7 times.
1: 200 times of dilution of test serum sample, every hole adds 100 μ l, and 37 ℃ of 1h discard liquid in the hole, clean 10 times with PBST.
Add enzyme and mark anti-IgG antibody 100 μ l, 37 ℃ of 1h discard liquid in the hole, clean 10 times with the PBST washing lotion.
Add TMD 100 μ l color development at room temperature 8min.
Add 50 μ l stop buffer cessation reactions.
Measure OD
450Result of determination.
Claims (10)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100535664C (en) * | 2006-06-28 | 2009-09-02 | 北京市农林科学院 | Detection method of aGST-ELISA of avian influenza virus (H5N1 subset) serum antibody |
| CN102495208A (en) * | 2011-10-24 | 2012-06-13 | 山东省农业科学院畜牧兽医研究所 | Method for diagnosing avian influenza virus by means of RT-PCR (reverse transcription-polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) and kit using method |
| CN104749372A (en) * | 2013-12-30 | 2015-07-01 | 北京义翘神州生物技术有限公司 | ELISA kit for H9N2 influenza virus hemagglutinin protein |
-
2005
- 2005-06-30 CN CN 200510027316 patent/CN1712965A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100535664C (en) * | 2006-06-28 | 2009-09-02 | 北京市农林科学院 | Detection method of aGST-ELISA of avian influenza virus (H5N1 subset) serum antibody |
| CN102495208A (en) * | 2011-10-24 | 2012-06-13 | 山东省农业科学院畜牧兽医研究所 | Method for diagnosing avian influenza virus by means of RT-PCR (reverse transcription-polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) and kit using method |
| CN102495208B (en) * | 2011-10-24 | 2014-03-26 | 山东省农业科学院畜牧兽医研究所 | Avian influenza virus RT-PCR (reverse transcription-polymerase chain reaction) kit |
| CN104749372A (en) * | 2013-12-30 | 2015-07-01 | 北京义翘神州生物技术有限公司 | ELISA kit for H9N2 influenza virus hemagglutinin protein |
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