CN105602907A - Infectious bronchitis virus resistance monoclonal antibodies - Google Patents

Abstract

The invention belongs to the field of immune detection of animal infectious diseases, in particular to an anti-chicken infectious bronchitis virus monoclonal antibody, the monoclonal antibody is secreted by a hybridoma cell line XYCDY-1BV-3G5, and the cell line is preserved in China Typical Culture Collection Center, the deposit number is CCTCC? NO: C201450. The other monoclonal antibody is secreted by the hybridoma cell line XYCDY-1BV-1E6 which is anti-chicken infectious bronchitis virus monoclonal antibody. NO: C201454. The hybridoma cell strain of the invention can be used in the preparation of monoclonal antibodies for detecting chicken infectious bronchitis virus antigens.

Description

A monoclonal antibody against chicken infectious bronchitis virus

The present invention is a divisional application of application number 2014100921707, and the filing date of the original application is March 13, 2014.

technical field

The invention belongs to the technical field of animal immunity application. Specifically relates to an anti-chicken infectious bronchitis virus monoclonal antibody, the monoclonal antibody can detect three kinds of poultry respiratory diseases (avian influenza H5 subtype and H9 subtype, Newcastle disease and chicken infectious bronchitis) pathogen detection triple Applied in rapid detection kits. The kit prepared by the invention can simultaneously complete the rapid detection of three poultry respiratory diseases such as avian influenza H5 subtype and H9 subtype, Newcastle disease and chicken infectious bronchitis pathogens at one time.

Background technique

Poultry respiratory infection is a very common disease in poultry production practice, especially the occurrence of this kind of disease is complex and changeable, which has brought huge economic losses to the poultry industry. The pathogens that cause the respiratory tract infection of poultry mainly include virus, bacterium and mycoplasma, wherein all are caused by RNA virus three kinds of diseases avian influenza (Avianinfluenza, AI), Newcastle disease (Newcastle disease, ND) and avian infectious bronchitis (Infectiouslaryngotrache, IB ) is more common in chicken farms. The three diseases have clinically similar respiratory symptoms. In addition, the clinical manifestations are mostly atypical, and the disease infection is mostly mixed. It is difficult to diagnose only based on clinical symptoms and pathological manifestations. Therefore, how to quickly and accurately make a diagnosis is an effective preventive treatment. Diseases are the root of avoiding serious economic losses.

At present, the methods for diagnosis of avian influenza, Newcastle disease and chicken infectious bronchitis are mainly molecular biological diagnosis and serological diagnosis. Molecular biology diagnostic techniques mainly include RT-PCR, nucleic acid probe technology, restriction enzyme fragment length polymorphism analysis, etc. Serological diagnosis mainly includes agar diffusion test (AGID), hemagglutination test and hemagglutination inhibition test (HA/HI), immunofluorescence, enzyme-linked immunoassay (ELISA) and immunochromatography. Molecular biological diagnostic methods have the characteristics of strong specificity and high sensitivity, but they have high requirements for experimental equipment and experimental personnel. Among serological diagnostic methods, agar diffusion test, hemagglutination test and hemagglutination inhibition test are relatively simple to operate, but their specificity and sensitivity are not high. Others such as immunofluorescence and enzyme-linked immunoassay also have high requirements for equipment, personnel and operation. Most of the above methods are time-consuming (such as 1.5-2 hours for RT-PCR; 24-48 hours for agar diffusion test; 2-3 hours for hemagglutination inhibition test; 3-5 hours for ELISA test, etc.), and it is not convenient to timely and rapid diagnosis, so neither can be widely used at the grassroots level.

Colloidal gold immunochromatography (gold-immunochromatography assay GICA) is a new type of immune labeling technology based on colloidal gold labeling technology applied to antigen-antibody reactions, using colloidal gold as a tracer. Its core technology uses nitrocellulose membrane as a solid phase carrier, and the sample solution is driven to swim on the chromatographic strip due to capillary action, and the analyte in the sample and the receptor for the analyte on the chromatographic membrane ( Such as antigen or antibody) produces highly specific and high affinity immune response in a short period of time. It has the advantages of simplicity, rapidity, strong specificity, high sensitivity and low cost.

Based on this technology, a variety of colloidal gold immunochromatographic rapid detection test cards have been developed in the fields of human and medical disease monitoring, environmental hazard monitoring, food safety assurance, and livestock and poultry disease detection. In the diagnosis and detection of chicken epidemic disease, the colloidal gold rapid detection kit for chicken Newcastle disease, chicken infectious bronchitis, chicken infectious bursa and mycoplasma gallisepticum antibody has obtained relevant patents (patent number: 200710169103.0; 200710169105.X; 200710169104.5; 200710169106.4).

But above-mentioned kit is to the detection of relevant disease or the antibody level after vaccine immunization, and can not accurately reflect whether chicken flock is infected with disease. Although there are also some immunocolloidal gold detection methods for a single pathogen at present, such as the "Avian Influenza Virus Antigen Detection Method and Its Gold Standard Rapid Diagnosis Kit and Preparation Method" with the patent application number 200510042631. "Influenza immune colloidal gold diagnostic test strips and test cards" discloses several preparation methods for detecting avian influenza virus antigens, but the diagnostic effect on mixed infection cases is not good, and multiple single-detection colloidal gold test strips are used for detection It also causes a waste of time and resources. Multivariate detection can detect multiple pathogens at one time, which solves this problem well. The ternary combined detection kit (test card) can reduce the consumption to one-third, thereby saving a lot of resources for the society and providing more sufficient time for effective and rapid treatment of diseases.

The present invention respectively prepares two strains of monoclonal antibodies against avian influenza, Newcastle disease and avian infectious bronchitis, uses double-antibody sandwich immunochromatography technology, and can detect whether a sample is avian influenza, Newcastle disease and Avian infectious bronchitis pathogen.

Contents of the invention

The purpose of the present invention is to overcome the defective of existing detection technology, provide a kind of anti-chicken infectious bronchitis virus monoclonal antibody, this monoclonal antibody can be in poultry three kinds of respiratory diseases (avian influenza H5 subtype and H9 subtype, Xincheng It is used in the triple rapid detection kit for the detection of pathogens of epidemic disease and chicken infectious bronchitis. The kit of the invention has strong specificity, high sensitivity and simple operation, and can be used to simultaneously complete the detection of three poultry respiratory disease pathogens of avian influenza H5 and H9 subtypes, Newcastle disease and chicken infectious bronchitis virus.

The invention also relates to the construction, development and utilization of six hybridoma cell lines secreting specific monoclonal antibodies.

The overall technical roadmap of the present invention is shown in Fig. 1 .

The present invention is realized through the following technical solutions:

In order to realize the present invention, the applicant has prepared two kinds of hybridoma cell lines XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7 which are directed at different antigenic sites and can be divided into anti-avian influenza virus (abbreviated as AIV). Virus (abbreviated as NDV) hybridoma cell lines XYCDY-NDV-3E11 and XYCDY-NDV-4B10 and two kinds of secretion resistant chicken infectious bronchitis virus (abbreviated as IBV) hybridoma cell lines XYCDY-1BV-3G5 and XYCDY-1BV-1E6 . The above-mentioned six cell lines were all sent to the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China for preservation on March 11, 2014, and the corresponding preservation information is summarized in Table 1.

Table 1 Preservation information of six hybridoma cell lines prepared by the present invention

On this basis, the applicant provides a test kit for triple rapid rapid avian influenza (H5 and H9 subtypes), Newcastle disease and chicken infectious bronchitis virus for three respiratory diseases of poultry, and the test kit includes a test card and a sample diluent , wherein the test card is composed of sample pad (1), gold standard pad (2), nitrocellulose membrane (3), absorbent pad (4), PVC backing (9) and test card shell (10), and its specific structure is : On the PVC backing (9), there are sample pads (1), gold standard pads (2), nitrocellulose membranes (3), and absorbent pads (4) adhered in sequence; the test strips are put into the test card case (10) constitute a test card. The gold-labeled pad (2) is coated with the three gold-labeled monoclonal antibody mixed solutions (the solution contains anti-avian influenza virus monoclonal antibody XYCSJL-AIV-2B8-colloidal gold marker, anti-Newtown Epidemic virus monoclonal antibody XYCDY-NDV-3E11-colloidal gold marker and anti-chicken infectious bronchitis virus monoclonal antibody XYCDY-1BV-3G5-colloidal gold marker); the nitrocellulose membrane (3) is wrapped The detection line T1 ( 5), T2(6) and T3(7); quality control line (8) contains rabbit anti-mouse IgG.

The applicant provides a method for rapidly preparing a triple rapid detection kit suitable for avian influenza H5 subtype and H9 subtype, Newcastle disease and chicken infectious bronchitis virus pathogens. The specific steps are as follows:

1) Avian influenza virus recombinant hemagglutinin HA1 protein (abbreviated as AIV-HA1), chicken Newcastle disease virus recombinant hemagglutinin HN protein (abbreviated as NDV-HN) and chicken infectious bronchitis virus recombinant nucleocapsid N protein (abbreviated as IBV-NP ) expression preparation. Three recombinant protein expression vectors pKG-HA1, pKG-HN and pKG-NP were constructed by cloning the sequence of avian influenza HA1 gene, Newcastle disease HN gene and chicken infectious bronchitis N gene and connecting the corresponding vectors. For the physical construction map of the expression vector pKG-HA1, please refer to the literature: China National Knowledge Network Dissertation Database: Wu Renwei, Research on avian influenza virus recombinant protein mucosal immunity and the establishment of N-ELISA antibody detection method [D] Huazhong Agricultural University, 2006http:// epub3.cnki.net/KCMS/detail/detail.aspx? dbcode＝CDFD&QueryID＝9&CurRec＝1&dbname＝CDFD9908&filename＝2006190169.nh&urlid＝&yx＝&uid＝WEEvREcwSlJHSldTTGJhYkhsd0Z6ODd6RU81ZVo0ODlGZ2hITVNLZlMwRHM5RzdZNG1mdWJmU3NZZFBoeTAwbg＝＝&v＝MTM0NThSOGVYMUx1eFlTN0RoMVQzcVRyV0。 The physical construction maps of the expression vectors pKG-HN and pKG-NP are shown in Figures 4 and 5 .

2) Purification of the three proteins AIV-HA1, NDV-HN and IBV-NP. The chromatographic column used for recombinant protein purification was GSTrap ^™ FFcolumns (purchased from GE Healthcare. USA). The recombinant protein was purified according to the instructions of GSTrap ^TM FFcolumns.

3) Balb/C mice (purchased from the Experimental Animal Center of Wuhan Institute of Biological Products) were immunized with the purified three recombinant proteins AIV-HA1, NDV-HN and IBV-NP respectively to obtain anti-avian influenza virus (AIV) hybrids Tumor cell lines XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7, anti-Newcastle disease virus (NDV) hybridoma cell lines XYCDY-NDV-3E11 and XYCDY-NDV-4B10, anti-chicken infectious bronchitis virus (IBV) hybridoma Cell lines XYCDY-1BV-3G5 and XYCDY-1BV-1E6 were injected into the treated Balb/C mice to obtain monoclonal antibody-containing ascites, which were subjected to octanoic acid-ammonium sulfate method ("Immunology Commonly Used" edited by Zhu Liping). Experimental Methods", People's Military Medical Publishing House, 2000 Edition) to obtain purified monoclonal antibodies. The six cell lines were all sent to China. Wuhan. Wuhan University China Type Culture Collection (CCTCC) on March 11, 2014. The preservation information is: hybridoma cell line XYCSJL-AIV-2B8, and its preservation number: CCTCCNO : C201449; hybridoma cell line XYCSJL-AIV-4B7, its preservation number: CCTCCNO: C201453; hybridoma cell line XYCDY-NDV-3E11, its preservation number: CCTCCNO: C201451; hybridoma cell line XYCDY-NDV-4B10, its Deposit number: CCTCCNO: C201455; hybridoma cell line XYCDY-IBV-3G5, its deposit number: CCTCCNO: C201450; hybridoma cell line XYCDY-IBV-1E6, its deposit number: CCTCCNO: C201454;

4) Extract mouse IgG and immunize healthy New Zealand rabbits (purchased from the Experimental Animal Center of Wuhan Institute of Biological Products) to obtain rabbit anti-mouse IgG antibodies;

5) Colloidal gold was prepared by reacting trisodium citrate with chloroauric acid (purchased from sigma company);

6) Add the anti-AIV antibody (XYCSJL-AIV-2B8), anti-NDV antibody (XYCDY-NDV-3E11) and anti-IBV antibody (XYCDY-1BV-3G5) prepared in step 3) to the colloidal gold prepared in step 5) , three kinds of monoclonal antibody-colloidal gold markers are mixed and concentrated according to a certain volume to form a gold-labeled mixture;

7) Coating the three gold standard mixtures on the gold standard pad (2);

8) Coat the anti-AIV antibody (XYCSJL-AIV-4B7), anti-NDV antibody (XYCDY-NDV-4B10) and anti-IBV antibody (XYCDY-1BV-1E6) prepared in step 3) on a nitrocellulose membrane (3 ) to form detection lines T1 (5), T2 (6) and T3 (7); and coat rabbit anti-mouse IgG on nitrocellulose membrane (3) to form quality control lines (8);

9) Adhering the sample pad (1), gold standard pad (2), nitrocellulose membrane (3), and absorbent pad (4) in sequence on the PVC backing (9) to obtain the Multiple detection immunocolloidal gold test strips for AIV (H5 or H9), NDV and IBV as described above.

10) Put the test strip described in 9) into the test card case (10) to form the test card.

Obviously, the present invention prepares six hybridoma cells that secrete specific monoclonal antibodies, which can be divided into three groups according to the same virus species and used as a double-antibody sandwich, wherein:

Hybridoma cells XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7 secreting anti-avian influenza virus monoclonal antibodies are used in pairs in the preparation and detection of avian influenza virus H5 and H9 subtype antigens.

Hybridoma cells XYCDY-NDV-3E11 and XYCDY-NDV-4B10 secreting monoclonal antibodies against Newcastle disease virus are used in pairs in the preparation and detection of Newcastle disease virus antigens.

Hybridoma cells XYCDY-1BV-3G5 and XYCDY-1BV-1E6 secreting monoclonal antibodies against chicken infectious bronchitis virus are used in pairs in the detection of Newcastle disease virus antigens.

Since the kit adopts the above-mentioned double-antibody sandwich method to coat the antibody, the detection effect of the kit of the present invention can be greatly improved.

The principle of the present invention is to adopt the double-antibody sandwich method (a conventional method for reporting), and to detect whether the sample contains avian influenza virus (AIVH5 or AIVH9) with the detection line T1 coated with anti-AIV antibody (XYCSJL-AIV-4B7) as an example. Explain that when the sample to be tested contains AIV (H5 or H9), the AIV (H5 or H9) in the sample will react with the gold-labeled anti-AIV antibody (XYCSJL-AIV-2B8) at the position of the gold label pad, and the reaction complex When the chromatography reaches the detection line position, the anti-AIV antibody coated on the detection line T1 will react with the AIV (H5 or H9) in the complex, thereby forming a "gold-labeled antibody-AIV-coated antibody" at the detection line position "Double-antibody sandwich complex, since one antibody in the complex is labeled with colloidal gold, a red band visible to the naked eye will appear at the detection line position, thus obtaining a positive result. The gold-labeled antibody that did not participate in the reaction was chromatographed to the position of the quality control line, and was captured by the coated rabbit anti-mouse IgG for color development. On the contrary, if there is no AIV (H5 or H9) in the sample, there is no double-antibody sandwich complex at the position of the detection line, and no red band will appear, thus a negative result is obtained. The detection test strip (structure diagram as shown in Figure 2) in the kit of the present invention consists of a sample pad (1), a gold standard pad (2), a nitrocellulose membrane (3), and an absorbent pad (4) according to the diagram. It is assembled sequentially by adhering on the PVC backing (9), and the test strips are packed into the test card case (10) to form the test card.

Compared with the prior art, the present invention has the following outstanding advantages:

1. The colloidal gold kit for multiple rapid detection of avian influenza H5 and H9 subtypes, Newcastle disease and chicken infectious bronchitis virus of the present invention has strong specificity, high sensitivity, short detection time (10-15 minutes), intuitive judgment, etc. advantage.

2. The kit of the present invention does not require any special instruments and equipment, and the detection cost is low. Simultaneously, because the invention realizes the simultaneous detection of the pathogens of three major poultry respiratory diseases (avian influenza H5 and H9 subtypes, Newcastle disease and chicken infectious bronchitis virus), the detection time and detection cost are more effectively saved.

3. The kit of the present invention is easy to operate.

4. The kit of the present invention is convenient to store, does not require high storage temperature, and has a shelf life of at least 90 days at 4°C.

Description of drawings

Figure 1: Overall technical roadmap of the present invention.

Figure 2: Schematic diagram of the assembly of the detection test strip of the present invention.

In the figure 1-sample pad, 2-gold standard pad, 3-nitrocellulose membrane, 4-absorbent pad, 5-test line T1, 6-test line T2, 7-test line T3, 8-quality control line, 9 -PVC backing, 10-test card case.

Fig. 3: Schematic diagram of judging the results of the test card of the present invention.

In the figure: 11: indicates that the detection of AIV, NDV, and IBV are all positive results; 12: indicates that the detection of AIV is positive, and both NDV and IBV are negative; 13: indicates that the detection of NDV is positive, and the results of AIV and IBV are negative; 14 : Indicates that IBV is positive, and AIV and NDV are both negative; 15: Indicates that AIV, NDV, and IBV are weakly positive; 16, 17: Indicates that the test card is invalid.

Fig. 4: The physical construction diagram of the recombinant protein expression plasmid related to NDV-HN in the present invention.

Fig. 5: A diagram of the physical construction of the recombinant protein expression plasmid related to IBV-NP of the present invention.

detailed description

Example 1

1. Preparation of AIV-HA1 protein, NDV-HN protein and IBV-NP protein

The preparation methods of AIV-HA1 protein, NDV-HN protein and IBV-NP protein mainly refer to the literature: Sambrook J, Fritsch EF, Mani Artis T (translated by Jin Dongyan, Li Mengfeng, etc.). Molecular Cloning Experiment Guide .Second Edition, Science Press, 1992 provided the method.

1.1 Cloning and sequencing of avian influenza virus HA1 gene and construction of expression vector pKG-NS1

The construction method and construction map of the avian influenza virus HA1 gene and its recombinant expression plasmid pKG-HA1 involved in the present invention have been published on CNKI in 2006, refer to the literature: CNKI Dissertation Library: Wu Renwei, Recombinant Protein of Avian Influenza Virus Mucosal immunity research and establishment of N-ELISA antibody detection method [D] Huazhong Agricultural University, 2006; see website: http://epub3.cnki.net/KCMS/detail/detail.aspx? dbcode＝CDFD&QueryID＝9&CurRec＝1&dbname＝CDFD9908&filename＝2006190169.nh&urlid＝&yx＝&uid＝WEEvREcwSlJHSldTTGJhYkhsd0Z6ODd6RU81ZVo0ODlGZ2hITVNLZlMwRHM5RzdZNG1mdWJmU3NZZFBoeTAwbg＝＝&v＝MTM0NThSOGVYMUx1eFlTN0RoMVQzcVRyV0；

1.2 Cloning and sequencing of Newcastle disease HN gene and construction of expression vector pKG-HN

The sequence and cloning method of the Newcastle disease HN gene involved in the present invention and the construction method of the expression vector pKG-HN The preliminary establishment of the method [D], Huazhong Agricultural University, 2006; see website: http://epub3.cnki.net/KCMS/detail/detail.aspx? dbcode＝CMFD&QueryID＝7&CurRec＝1&dbname＝CMFD9908&filename＝2006190369.nh&urlid＝&yx＝&uid＝WEEvREcwSlJHSldTTGJhYlNcGZ2OHlzRkwxL0k2RVBNdlRoYWMzMllid1d5bUVYOXE3ZU9jcm11MC91WmJUeA＝＝&v＝MjI3OTh6TVYxMjdHTEt4SHRMS3BwRWJQSVI。

The specific implementation method is as follows: inoculate NDV in the allantoic cavity with a strain of Newcastle disease NDV LaSota strain preserved in the Microbiology and Immunity Laboratory of Huazhong Agricultural University, and inoculate NDV at the age of 9-11 days in healthy SPF (specific pathogen-free) chicken embryos (purchased from Wuhan Institute of Biological Products), discard dead embryos 24 hours ago, collect chicken embryo allantoic fluid 24-96 hours; centrifuge at 4,000rpm for 30min at 4°C, take the supernatant; centrifuge at 30,000rpm for 60min at 4°C to concentrate the virus. The precipitate was dissolved with PBS (prepared with DEPC-treated water) at pH 7.2, and stored at -70°C after aliquoting. Primers were designed and synthesized according to the HN gene sequence of NDV LaSota isolates collected in GenBank (accession number: AJ629060.1) to amplify the HN gene. Upstream primer P1: ATA GGATCC GGGGCTAGCACACTTA; downstream primer P2: AGC AAGCTT CTAGCCAGACTTGGCT. P1 is located upstream of the promoter, with a BamHI site added, and P2 contains a stop codon, with a HindIII site added, and the two restriction sites are underlined. Between the two primers is the HN gene that lacks the sequences of the cytoplasmic region and the transmembrane region. Take an appropriate amount of virus suspension to extract RNA, amplify DNA according to the method provided in the TaKaRaRNAPCRKit (AMV) Ver. The recovered RT-PCR product was directly ligated with the cloned pMD18-T purchased from TaKaRa Company, and then the ligated product was transformed into the competent cell Escherichia coli DH5α in the kit (commercial kit purchased from TaKaRa Company), Pick positive clones and use alkaline lysis method (Sambrook J, Fritsch EF, Maniartis T editors (translated by Jin Dongyan, Li Mengfeng, etc.). Molecular Cloning Experiment Guide. Second Edition, Beijing Science Press, 1992 version) to extract the recombinant plasmid DNA and identify the recombinant plasmid DNA. The correctly identified recombinant plasmid was named pMT-HN, and sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing, and then the sequence determination results were compared and analyzed for homology with the relevant data in GeneBank.

pMT-HN was digested with BamHI and HindIII, and a fragment of about 1590pb was recovered, and then this fragment was ligated with the expression vector pGEX-KG of Amersham Biosciences, which was digested with the same enzyme, to construct an expression vector. The recombinant expression vector was transformed into freshly prepared DH5α competent cells, and the transformed bacteria were coated with ampicillin-containing LB agar plates and cultured overnight at 37°C. After picking a few single colonies and culturing them overnight, a small amount of plasmids were prepared by alkaline lysis method, and then analyzed by enzyme digestion and identified by PCR amplification. The obtained positive clone was named pKG-HN. A large amount of plasmid DNA was prepared by alkaline lysis method for the positive clones, and frozen at -20°C. The construction process of the recombinant expression plasmid pKG-HN is shown in Figure 4.

1.3 Cloning and sequencing of chicken infectious bronchitis N gene and construction of expression vector pKG-NP

The sequence and cloning method of the N gene of Newcastle disease and chicken infectious bronchitis involved in the present invention and the construction method of the expression vector pKG-NP Cloning and expression of nucleoprotein gene and its preliminary application in antibody detection [D], Huazhong Agricultural University, 2006; see website: http://epub3.cnki.net/KCMS/detail/detail.aspx? dbcode＝CMFD&QueryID＝14&CurRec＝1&dbname＝CMFD9908&filename＝2006190367.nh&urlid＝&yx＝&uid＝WEEvREcwSlJHSldTTGJhYlNPcGZ2OHlzRkwxL0k2RVBNdlRoYWMzMllid1d5bUVYOXE3ZU9jcm11MC91WmJUeA＝＝&v＝MjQ5NDY2ZlpPZHVGeXpnVnJ6SVYxMjdHT。 The specific implementation method is as follows: a strain of chicken infectious bronchitis IBV (H52 strain) strain preserved by freeze-drying in the Veterinary Microbiology and Immunity Laboratory of Huazhong Agricultural University is used to inoculate NDV in the allantoic cavity at the age of 9-11 days with healthy SPF (specific pathogen free) ) chicken embryos (purchased from Wuhan Institute of Biological Products), discard the dead embryos 24 hours ago, and collect the allantoic fluid of chicken embryos 24-96 hours; centrifuge at 4,000rpm for 30min at 4°C, and take the supernatant; 30,000 at 4°C The virus was concentrated by centrifugation at rpm for 60 min. The precipitate was dissolved with PBS (prepared with DEPC-treated water) at pH 7.2, and stored at -70°C after aliquoting. According to the N gene sequence of IBV recorded in GenBank (accession number: EF602459.1), the more conservative sequence in the N gene was designed and synthesized to amplify the N gene. Upstream primer P1: GGATCC ATGGCAAGCGGTAAAGCAG; downstream primer P2: CC GAGCTC TCAAAGTTCATTCTCTCC. P1 is located upstream of the promoter, with a BamHI point added, and P2 contains a stop codon, with a ScaI site added, and the two restriction sites are underlined. Between the two primers is a relatively conserved fragment in the N gene. Take an appropriate amount of virus suspension to extract RNA, amplify DNA according to the method provided in the TaKaRaRNAPCRKit (AMV) Ver. The recovered RT-PCR product was directly ligated with the cloned pMD18-T purchased from TaKaRa Company, and then the ligated product was transformed into the competent cell Escherichia coli DH5α in the kit (commercial kit purchased from TaKaRa Company), Pick positive clones and use alkaline lysis method (Sambrook J, Fritsch EF, Maniartis T editors (translated by Jin Dongyan, Li Mengfeng, etc.). Molecular Cloning Experiment Guide. Second Edition, Beijing Science Press, 1992 version) to extract the recombinant plasmid DNA and identify the recombinant plasmid DNA. The correctly identified recombinant plasmid was named pMT-N, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and then the sequence determination results were compared and analyzed for homology with the relevant data in GeneBank.

pMT-N was digested with BamHI and ScaI, and a fragment of about 1.2 kb was recovered, and then this fragment was ligated with the expression vector pGEX-KG of Amersham Biosciences Company digested with the same enzyme to construct an expression vector. The recombinant expression vector was transformed into freshly prepared DH5α competent cells, and the transformed bacteria were coated with ampicillin-containing LB agar plates and cultured overnight at 37°C. After picking a few single colonies and culturing them overnight, a small amount of plasmids were prepared by alkaline lysis method, and then analyzed by enzyme digestion and identified by PCR amplification. The obtained positive clone was named pKG-NP. A large amount of plasmid DNA was prepared by alkaline lysis method for the positive clones, and frozen at -20°C. The construction process of the recombinant expression plasmid pKG-NP is shown in Figure 5.

1.4 Expression of three proteins AIV-HA1, NDV-HN and IBV-NP

Transform Escherichia coli BL21 (DE3) with recombinant plasmids pKG-HA1, pKG-HN and pKG-NP and expression vector pGEX-KG, respectively, and coat LB plates containing ampicillin (Amp) (1% (W/V) Tryptone, 0.5 %(W/V) YeastExtract, 0.5% (W/V) NaCl, 0.1mg/mLAmp and 1.5% (W/V) Agar), cultured in a 37°C incubator, picked a single colony, and inoculated it in 2.0mL containing ampicillin Penicillin (Amp) liquid LB medium (1% (W/V) Tryptone; 0.5% (W/V) YeastExtract; 0.5% (W/V) NaCl and 0.1mg/mL Amp), 37 ° C 250-300r/ Shake culture for min, when OD600nm reaches 0.6-1.0. Inoculate 1.0 mL of the bacterial solution into 10.0 mL of liquid LB medium containing ampicillin (Amp), culture at 37°C for 3 h with shaking at 250-300 r/min, and then add the inducer IPTG (isopropyl -β-D-thiogalactopyranoside), induced for 4 hours, and the cells were collected by centrifugation for SDS-PAGE detection.

1.5 Purification of three proteins AIV-HA1, NDV-HN and IBV-NP

(1) Purification of supernatant

A large amount of expression was induced according to the conditions in the above 1.4, the induced bacterial culture was centrifuged at 8,000r/min for 10min, and PBS (phosphate) buffer (140mMNaCl, 2.4mMKCl, 10mMNa2HPO4.12H2O, 1.8mMKH2PO4pH7.4) was used for precipitation ( About 1/10 of the volume of the original bacterial solution), add DTT (dithiothreitol) to a final concentration of 10mmol/L, crush it with a pressure breaker until the liquid becomes transparent, and centrifuge at 12,000r/min at 4°C After 15 minutes, the crushed supernatant was filtered with a 0.22 μm filter membrane, and the above supernatant was purified by the AKTA protein purification system AKTAexplorer 10 (purchased from GE Healthcare, USA) with a GSTrapTMFFcolumns chromatography column.

(2) Purification and renaturation of inclusion bodies

Wash the crushed and centrifuged precipitate twice with PBS (pH7.4) in the above (1), add 19.7 mL of buffer A (50 mM Tris-Cl, 0.5 mM EDTA, 50 mM NaCl, 5% (W/V) Glycerol) and 0.3 mL of 20 % (W/V) SKL (sodium lauryl sarcosinate) stock solution, stir vigorously to dissolve, let it stand for 2 hours, centrifuge at 12000r/min at 4°C for 10 minutes, discard the precipitate, take the supernatant, add 20% (W/V) PEG-4000 (polyethylene glycol 4000) to a final concentration of 0.2%, add 50mmol/L oxidized glutathione to a final concentration of 1mmol/L, add 100mmol/L reduced glutathione The glycosides were left to stand for 2 hours to a final concentration of 2 mmol/L, dialyzed against PBS (pH 7.4) for 2 to 3 days, and stored at -70°C for later use.

(3) Determination and identification of protein concentration

The light absorption values of the protein solution at 280nm and 260nm wavelengths were respectively measured with a nucleic acid protein analyzer to A280 and A260. Calculate the protein concentration according to the formula, divide the protein solution that reaches the required concentration into 100 μL/tube, take 50 μL for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) identification and Western-blot analysis, and the rest Store at -70°C for later use.

SDS-PAGE detection: Take the collected bacteria, fully resuspend the bacteria with 40.0 μL ddH2O, add an equal volume of sample buffer (250mM Tris-HCl, 10% (W/V) SDS, 0.5% (W/V) Bromophenol blue, 50% (V/V) glycerol, 5% (V/V) β-mercaptoethanol), boiled in a water bath for 3-5min, and then waited for sample electrophoresis in an ice bath. According to Sam Brook J, Fritsch EF, editor-in-chief of Manny Attis T; Jin Dongyan, Li Mengfeng etc. translation, "Molecular Cloning Experiment Guide", the second edition, the method described in the 1992 edition to prepare separating gel and stacking gel, add 1 × Tris-glycine electrophoresis buffer (25mM Tris-base, 0.1% (W/V) SDS, 0.25MGlycine), take the processed sample spotting, electrophoresis 2h, remove the gel, Coomassie brilliant blue R250 staining solution (0.1% (W/V) Coomassie Brilliant Blue R250, 25% (V/V) isopropanol, 10% (V/V) glacial acetic acid) stained for 2h, and then destained with SDS-PAGE solution (5% (V/V) ethanol , 10% (V/V) glacial acetic acid) for decolorization, observed and photographed. And the expressed foreign protein content was analyzed by gel thin layer scanning.

Western-blot analysis: In order to analyze the immunological activity of the expressed fusion proteins AIV-HA1, NDV-HN and IBV-NP with GST tags, SDS-PAGE electrophoresis was carried out as described above, and the gel after electrophoresis was not After staining, the protein was transferred directly to the NC membrane with a transfer device, and was electrotransferred at 15V for 1.5h. After the transfer, rinse the NC membrane in TBST (150mMNaCl, 20mM Tris-HCl, 0.05% Tween20), then transfer the NC membrane into a heat-sealable plastic bag, press 0.1mL-0.15mL/cm2 (NC membrane area) into the blocking solution (TBST containing 1% (W/V) bovine serum albumin), after exhausting the air bubbles in the bag as much as possible, heat and seal the bag mouth, shake gently at room temperature for 1-2h or overnight at 4°C. Then discard the blocking solution, take out the membrane, transfer the membrane into a new plastic bag, add chicken anti-AIV hyperimmune serum diluted with TBST (1:150) in an amount of 0.1mL-0.15mL/cm2, and remove air bubbles as above. Seal the mouth of the bag, act on a shaker at room temperature for 1 hour, wash 6 times with TBST, 5mim each time, transfer the membrane to a new plastic bag, add rabbit antibody diluted with TBST (1:5000) at 0.1mL-0.15mL/cm2 Chicken IgG-HRP (purchased from GenScript Company), reacted at room temperature for 1 h, washed 6 times with TBST, 5 min each time, and finally rinsed 2 times with TBS, added the substrate solution prepared with 0.01 mol/LTris-Cl (pH 7.6) (DAB-H2O2) color development, once the protein band appears, stop it with ddH ₂ O immediately, then observe and take pictures.

According to the formula calculation (protein concentration (mg/mL)=1.45*A280-0.74*A260), the target protein concentrations obtained for purification are: HA1--0.45mg/mL; HN--0.53mg/mL; N-- 0.90mg/mL. Utilize Western-blot analysis result to show that the recombinant AIV-HA1 protein (62KDa) of the present invention, NDV-HN protein (84KDa), IBV-NP protein (72 and 60KDa) have reacted specifically with corresponding positive serum respectively, confirm These kinds of expression products all have good antigenicity.

2. Preparation of anti-AIV antibody, anti-NDV antibody and anti-IBV antibody:

The three recombinant proteins AIV-HA1, NDV-HN and IBV-NP prepared by the applicant were used to immunize Balb/C mice (purchased from the Experimental Animal Center of Wuhan Institute of Biological Products), and the immunization procedure was to take a solution with a protein content of 100 μg Emulsified with an equal volume of Freund's complete adjuvant (purchased from sigma company), injected into the abdominal cavity of mice, boosted immunization 21 days later, and emulsified with Freund's incomplete adjuvant (purchased from sigma company), and finally three days before fusion, (preferably more than 4 weeks apart from the last immunization), intraperitoneal booster immunization, double the amount of antigen (200μg), without adjuvant. At the time of fusion, one Balb/C mouse that had undergone the final booster immunization was taken, sacrificed by bleeding from the orbit (collecting serum, it was positive serum), and sterilized by soaking in 75% alcohol for 5 minutes. Aseptically remove the spleen of the mouse, separate the splenocytes, and the resuscitated SP2/0 myeloma cells (SP2/0 myeloma cells are donated by the Wuhan Institute of Biological Products, Ministry of Health) by 1-2×107 SP2/0 and 108 The ratio of immune cells (1:10 to 1:15) was mixed in a 50mL centrifuge tube, and centrifuged at 1500rpm for 10min. Pour off the supernatant (use sterilized filter paper to blot dry), tap the bottom of the tube gently to loosen the cell pellet slightly. Place the centrifuge tube containing the cell mixture in a 37°C water bath. Then, 0.8 mL of 50% PEG (purchased from sigma company) pre-warmed to 37° C. was slowly added dropwise within 1 min, stirred gently with a pipette tip while adding, and continued to stir for 1 min. Then slowly add 10 mL of RPMI-1640 basal culture medium (purchased from GIBCO) pre-warmed at 37°C. The specific method is: add 1mL drop by drop in the first minute, add 1ml in the second minute, add 3mL in the 3rd to 4th minute, add the remaining 5mL in the 5th minute, add slowly each time, and keep stirring gently. Finally, add 30mL of 1640 solution, also need to add slowly. Centrifuge at 800rpm for 5min, remove the supernatant, and place at 37°C for 5-8min. Suspend with HAT (purchased from GIBCO company) medium, and also use HAT medium to suspend the prepared feeder splenocytes and mix with the fused cells, and add an appropriate amount of HAT medium as needed (composition of the medium: 80 mL of RPMI-1640 basal culture medium, 20mL sterilized calf serum, 1mL 100% HAT, 1mL 10,000U/mL penicillin-streptomycin double antibody), in 96-well culture plate, about 250μL/well. One fusion can inoculate 4-8 96-well plates. According to the needs, it can also be less. Generally, the number of SP2/0 myeloma cells is calculated, and the inoculation amount of each well contains about 104 SP2/0 myeloma cells. Cultivate in a 37°C, 5% CO2 incubator. On the second day after fusion, observe whether there is any contamination, add 1 drop of HAT medium on the 4th day, absorb 100 μL of the medium on the 8th to 10th day and replace it with 100 μL of HT (purchased from GIBCO Company) medium. When the fused cell colony grows to 1/4 of the culture well and the medium turns slightly yellow, carry out antibody detection. For the AI fusion hole, AIV-HA1 fusion protein and AIV allantoic fluid were used as the coating source, the ND fusion hole was coated with NDV-HN fusion protein and NDV allantoic fluid, and the IB fusion hole was coated with IBV-NP fusion and IBV allantoic fluid, both were detected by double screening, and the positive wells secreting corresponding monoclonal antibodies were screened out by conventional ELISA method. The screened positive wells were immediately cloned and screened by the limiting dilution method (refer to Xue Qingshan, "Principles and Techniques of In Vitro Culture", Science Press, 2001 edition). After 3 to 4 times of cloning, the hybridoma cells XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7 secreting anti-avian influenza virus (AIV) antibodies were finally screened out, and the hybridoma cells XYCDY-NDV- 3E11 and XYCDY-NDV-4B10 and anti-chicken infectious bronchitis virus (IBV) antibody hybridoma cells XYCDY-1BV-3G5 and XYCDY-1BV-1E6 a total of 6 strains of cells (see the "Content of the Invention" part of this description for biological deposit information ). Chromosome counting was carried out on the above 6 cell lines, and the results showed that the average number of chromosomes of SP2/0 myeloma cells was 70, the number of chromosomes of splenocytes was 40, and the number of chromosomes of hybridoma cells was between 80 and 94. Both were higher than the number of chromosomes of the two parental cells, indicating that the fusion cell was indeed a hybrid product of SP2/0 myeloma cells and splenocytes, and the number of chromosomes of hybridoma cells was significantly more than that of SP2/0 myeloma cells. Two cell lines against the same pathogen were identified by additive ELISA as producing antibodies against different epitopes. Six cell lines (1×10 6 ) of the present invention were injected into the peritoneal cavity of Balb/C mice respectively to produce monoclonal antibodies. The mouse Mab Isotyping Test Kit (Mouse Mab Isotyping Test Kit) from ROCKLAND Company was used to identify the subtype of the monoclonal antibody obtained in the present invention, and the results were all of the mouse IgG2b subtype.

3. Purification of Monoclonal Antibodies

With reference to Zhu Liping, "commonly used experimental methods in immunology", the method reported in the People's Military Medical Press, 2000 edition: get the mouse ascites 5mL of gain and mix with an appropriate amount of silicon dioxide, add an equal volume of barbiturate buffer ( Recipe: sodium chloride 85.00g, barbiturate 5.75g, barbital sodium 3.75g, sodium azide 2.00g, dilute to 2000mL with distilled water), shake at room temperature for 1h, let stand at room temperature for 30min, take the supernatant In a clean centrifuge tube, centrifuge at 4°C, 3000rpm for 10min; take 8mL of the supernatant, add 16mL of 0.06mol sodium acetate buffer, adjust the pH value to 4.5 with HCl, slowly add 132μL of caprylic acid under full stirring, and stir at room temperature for 30min , and then transferred to 4°C refrigerator for sufficient precipitation for 2h, centrifuged at 15000rpm for 30min at 4°C to obtain 22mL of supernatant, added 2.2mL of 0.1M phosphate buffer (referred to as PB, formula: 10mMNa ₂ HPO ₄ .12H ₂ O and 1.8mM KH ₂ PO ₄ pH7.2), adjust the pH value to 7.6 with NaOH, slowly add ammonium sulfate under stirring to a final concentration of 0.277g/mL, fully precipitate in the refrigerator at 4°C for 2h, centrifuge at 12000rpm for 30min at 4°C, discard The precipitate was resuspended with 5mL of 0.01M PBS buffer solution (recipe: 140mMNaCl, 2.4mMKCl, 10mMNa ₂ HPO ₄ .12H ₂ O, 1.8mMKH ₂ PO ₄ pH7.2), loaded into a dialysis bag, and used for 5000mL 0.01MpH7. 2 After the PBS buffer is fully dialyzed, dialyze against 2000mL distilled water, and finally dialyze against 3000mL triple-distilled deionized water, concentrate the fully dialyzed protein solution to 3mL with PEG-20000, then centrifuge at 4°C and 12000rpm for 30min, discard the precipitate, The supernatant was collected, and the concentration of the six monoclonal antibodies was measured to be between 1.0-2.2 mg/mL. The purified monoclonal antibody identified by SDS-PAGE has a purity greater than 95%. The monoclonal antibody can be used to prepare immune colloidal gold.

4. Preparation of rabbit anti-mouse IgG antibody:

Using Balb/C mouse IgG to immunize healthy New Zealand white rabbits, prepare rabbit anti-mouse IgG hyperimmune serum with high specificity and high titer, and use saturated ammonium sulfate precipitation method for hyperimmune serum (reference: Zhu Liping, Immunology Commonly Used Experimental Methods", People's Military Medical Press, 2000 edition) for rough extraction, and after passing through the G-200 column, high-purity rabbit anti-mouse IgG antibody was obtained. We use this antibody as the core reagent of the quality control line of the kit.

5. Preparation of monoclonal antibody-colloidal gold label

(1) Preparation of colloidal gold:

Dilute 1% chloroauric acid to 0.01% with double-distilled deionized water, stir and boil on a magnetic heating stirrer, add 1.5mL 1% trisodium citrate for every 100mL of 0.01% chloroauric acid, and continue to boil until the liquid is Stop heating when orange-red, and make up for water loss after cooling to room temperature. The appearance of the prepared colloidal gold should be pure, translucent, free of precipitation and floating matter, and stored at 4°C.

(2) Preparation of monoclonal antibody-colloidal gold marker:

The obtained anti-AIV antibody XYCSJL-AIV-2B8, anti-NDV antibody XYCDY-NDV-3E11 and anti-IBV antibody XYCDY-1BV-3G5 were respectively labeled with colloidal gold particles. The specific steps are as follows: under magnetic stirring, adjust the pH value of colloidal gold to 8.0 with 0.1M potassium carbonate solution, and add the above three monoclonal antibodies (anti-AIV antibody XYCSJL-AIV-2B8) at 5-7.2 μg antibody/mL colloidal gold , anti-NDV antibody XYCDY-NDV-3E11 and anti-IBV antibody XYCDY-1BV-3G5), continue to stir and mix for 30 minutes, add 10% BSA (bovine serum albumin) to a final concentration of 1%, and let stand for 30 minutes. Centrifuge at 12000rpm at 4°C for 30min, discard the supernatant, and wash the precipitate with 0.02M pH9.0 borate buffer (recipe: 0.1237g boric acid, PEG-200001g, dilute to 1000mL with triple distilled water, adjust pH to 9.0) for two washes. Once, use 0.02MpH9.0 borate buffer (recipe: boric acid 0.1237g, PEG-200001g, dilute to 1000mL with three distilled water, adjust pH to 9.0) with 0.02MpH9.0 borate buffer solution of 1/20 initial volume of colloidal gold Resuspend and store at 4°C for later use, with a shelf life of 60 days.

6. Coating of gold standard pad

Soak the gold standard pad in blocking solution (recipe: 2% BSA, 2.5% sucrose, 0.3% PVPK-30, 0.02% NaN3, 0.5% Teewn-20, 0.5% PEG-2000, 10mMNa ₂ HPO ₄ .12H ₂ O and 1.8mMKH ₂ PO ₄ pH 7.2) for 30min, then dried at 37°C. Take equal volumes of the above-mentioned three kinds of monoclonal antibodies prepared above (anti-AIV antibody XYCSJL-AIV-2B8, anti-NDV antibody XYCDY-NDV-3E11 and anti-IBV antibody XYCDY-1BV-3G5) Antibody gold standard substance is fully mixed and concentrated, Coat the prepared monoclonal antibody-colloidal gold marker evenly on the gold label pad with a Biodot dot film instrument, coat 9 μL of the antibody-colloidal gold marker per centimeter of the gold label pad, vacuum dry (according to conventional methods), and vacuum seal (According to the conventional method), put it at 4°C for later use.

7. Handling of the sample pad

Soak the sample pad in blocking solution (containing 2% BSA, 2.5% sucrose, 0.3% PVPK-30, 0.02% NaN3, 0.5% Teewn-20, 0.5% PEG-2000, 10mMNa ₂ HPO ₄ .12H ₂ O and 1.8mMKH ₂ PO _{4 (} pH6.4) for 30 minutes, dried at 37°C, sealed in vacuum, and placed at 4°C for later use.

8. Coating of Nitrocellulose Membrane

Anti-AIV antibody XYCSJL-AIV-4B7, anti-NDV antibody XYCDY-NDV-4B10 and anti-IBV antibody XYCDY-1BV-1E6 were diluted 10 with coating solution (0.01M pH7.4PBS buffer containing 3% methanol, 1% sucrose) -18μg/mL, it was coated on the nitrocellulose membrane successively with Biodot film spotting instrument as the detection line (the detection line was T1, T2 and T3 in turn), the coating volume was 0.7μL/cm, and the detection line was close to the gold The pad end of the standard pad is about 5mm away from the pad end of the gold standard pad; dilute the rabbit anti-mouse IgG antibody to 500μg/mL with the coating solution, and coat it on the nitrocellulose membrane with a Biodot membrane analyzer as a quality control line. The volume is 0.7μL/cm, the quality control line is close to the absorbent pad, about 5mm away from the absorbent pad, and the distance between two quality control lines or detection lines is 3-4mm. Dry at 37°C for 30-40min and set aside.

9. Assembly of the Kit

Adhere the sample pad (1), gold standard pad (2), nitrocellulose membrane (3), and absorbent pad (4) on the PVC backing (7) in the order shown in Figure 2, and cut them into 4mm wide The strips are packaged in the test card case (10) to form a test card, and after the preparation is completed, the test card in the kit is vacuum-packed. Stored at 4°C, the shelf life is at least 90 days.

Embodiment 2 (application embodiment)

Method of using triple rapid detection kit for poultry major respiratory diseases

1. The composition of the kit, including:

① 25 test cards

②One bottle of sample diluent (10mL/bottle)

2. Sample Preparation

2.1 Sample diluent: The sample diluent is 0.85% sodium chloride solution. Preparation method: 8.5gNaCl, add distilled water to make up to 1000mL.

2.2 Sample preparation

The detailed operation method of sample collection and processing is as follows:

(1) Sample of trachea and pharynx: First, remove the chicken's howl by hand, then pull out the chicken's tongue by hand or tweezers to expose the pharynx, insert a cotton swab into the trachea of the pharynx, stir it a few times, take it out, and then Put it into the sample tube that has been added with 500 μL of sample diluent in advance, stir and squeeze vigorously to elute the sample on the cotton swab as much as possible, and after standing for 15 minutes, the liquid supernatant in the tube is the sample to be tested;

(2) Cloacal sample or fecal stain sample on the poultry transport vehicle: directly use a cotton swab to take a sample from the chicken's cloaca or dip the feces stains on the poultry transport vehicle, and then put the cotton swab into the 500μL sample diluent In the sample tube, elute the sample as above, discard the cotton swab, and the supernatant in the tube after standing still is the sample to be tested;

(3) Muscle or visceral samples: Take a small piece of chest muscle or thigh muscle or a small piece of internal organs (1-2g), add 1-3mL sample diluent to the grinder and grind thoroughly, freeze-thaw once, 4,000r /min, centrifuge for 5min, and the supernatant is the sample to be tested.

3. Detection: Take out the kit, equilibrate at room temperature for 20 minutes; open the packaging bag, take out the test card, take 100-120 μL of the prepared sample and drop it into the sample hole of the test card, and judge the result within 10-15 minutes.

4. Result judgment: As shown in Figure 4, the detection lines T1, T2 and T3 on the NC membrane correspond to the detection of AIVH5 or AIVH9, NDV and IBV, respectively. When the quality control line on the NC membrane in the test card appears purplish red visible to the naked eye, but the corresponding detection line such as T1 (or T2 or T3) does not appear purplish red visible to the naked eye, the test sample does not contain AIVH5 or AIVH9 (or NDV or IBV), the result is judged as negative and recorded as "-"; when the test card shows a purple-red quality control line visible to the naked eye, and the corresponding detection line such as T1 (or T2 or T3) appears purple-red visible to the naked eye, If the test sample contains AIVH5 or AIVH9 (NDV or IBV), the result is judged as positive and recorded as "+"; the color depth of the test line is positively correlated with the virus content in the tested serum, and the darker the color, the higher the virus content in the tested sample. High, if there is no band on the quality control line, it is judged that the detection test card is invalid.

Example 3

1 Example of the application of the triple rapid detection kit for major poultry respiratory diseases

1.1 Detection of Chicken Standard Antigen

①Specificity test: Commercially purchased avian influenza H5 subtype HI antigen, avian influenza H9 subtype HI antigen, chicken Newcastle disease HI antigen, infectious bronchitis HI antigen (the above biological materials were purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) , Egg Drop Syndrome (EDSV-76) HI Antigen, Infectious Bursal of Fabricius (IBD) Agar-Extended Antigen, Mycoplasma Gallisepticum Antigen, Chicken Infectious Laryngotracheitis (AILT) Virus Antigen (the above biological materials were purchased from Beijing . China Veterinary Drug Administration), 0.85% sodium chloride solution and negative allantoic fluid were tested according to the method described in Example 2 of the present invention (GICA). When the detection test card in the kit of the present invention detects avian influenza H5 subtype HI antigen, avian influenza H9 subtype HI antigen, Newcastle disease HI antigen, infectious bronchitis HI antigen, the quality control line and the corresponding detection line all appear obvious Purple-red bands, and non-corresponding detection lines will not show purple-red bands (for example, when detecting avian influenza H5 subtype HI antigen, the quality control line and the detection line T1 appear purple-red bands at the same time, while the detection lines T2 and T3 do not appear. Bands appear; while detecting 3 kinds of above-mentioned antigens at the same time, the quality control line and detection line T1, T2 and T3 simultaneously appear purple-red bands); while detecting 0.85% sodium chloride solution (blank), negative allantoic fluid, minus Egg syndrome (EDSV-76) HI antigen, chicken infectious bursa (IBD) agar expansion antigen, mycoplasma gallisepticum antigen and chicken infectious laryngotracheitis virus antigen, only the quality control line appeared purple band. This shows that the specificity of the test card in the kit of the present invention is high, and there is no cross-reaction with other important disease antigens of poultry.

Table 2 applies test card of the present invention to poultry standard antigen-specific detection result

Note: "+" indicates that the detection line develops color, and "-" indicates that the detection line does not develop color.

②Sensitivity test: use 0.85% sodium chloride solution to avian influenza H5 (H9) subtype HI antigen, chicken Newcastle disease HI antigen, infectious bronchitis HI antigen respectively first diluted 10 times and then multiple dilution, the final dilution is 1: 10240, take 100-120 μ L of diluted samples to test according to the kit test card of the present invention. The results are shown in Table 3. When the avian influenza H5 (or H9) subtype HI antigen was diluted to 1:2560, the chicken Newcastle disease HI antigen was diluted to 1:1280, and the infectious bronchitis HI antigen was diluted to 1:640, the test card still showed A positive reaction indicates that the test card in this kit has high sensitivity. Prepare AIV, NDV and IBV three pathogenic single detection test cards to test its sensitivity at the same time, the result shows that the test card in the kit of the present invention has no difference with the three kinds of pathogenic single detection test cards.

Table 3 test card of the present invention detects the sensitivity test result of three kinds of standard antigens

1.2 The kit of the present invention detects the sample sent for inspection and compares it with the virus isolation and detection method

118 parts of tracheal swab samples were collected from chicken farms around Wuhan City, Hubei Province with the test kit of the present invention (see Example 2 for the preparation of samples), and virus isolation and identification were carried out to the samples at the same time, with hemagglutination (HA ) and the detection of hemagglutination inhibition (HI) as a control, wherein the detection of AIV refers to the national standard GB/T18936-2003 of the People's Republic of China, the detection of NDV refers to the national standard GB/T16550-2008 of the People's Republic of China, and the detection of IBV refers to the national standard GB/T16550-2008 of the People's Republic of China /T23197-2008. The detection result of comparison between the two is shown in Table 4-6, as can be seen from Table 4, when detecting AIVH5 (or H9) subtype, the positive coincidence rate of test kit of the present invention and HA and HI detection is 70% and The negative coincidence rate was 90.7%, and the total coincidence rate was 92.3%. As can be seen from Table 5, when detecting NDV, the positive coincidence rate of the kit of the present invention and HA and HI detection is 76.5%, the negative coincidence rate is 91.7%, and the total coincidence rate is 96.2%. And as can be seen from Table 6, when detecting IBV, the positive coincidence rate of the kit of the present invention and HA and HI detection is 70.6% and the negative coincidence rate is 95.3%, and the total coincidence rate is 95.7%. The results show that the detection results of the kit of the present invention are basically consistent with those of the HA and HI detection methods, and the coincidence rate is good.

The clinical detection AIV result of test kit of the present invention and hemagglutination (HA) and hemagglutination inhibition (HI) detection of table 4

The clinical detection NDV result of test kit of the present invention and hemagglutination (HA) and hemagglutination inhibition (HI) detection of table 5

The clinical detection IBV result of test kit of the present invention and hemagglutination (HA) and hemagglutination inhibition (HI) detection of table 6

Example 4

Evaluation of the Stability of the Triple Rapid Detection Kit for Three Poultry Respiratory Diseases of the Present Invention

The vacuum-packaged kit test cards of the present invention placed at 4°C and 37°C respectively were taken out on the 7th, 16th, 35th, 54th, 70th, and 90th day, and tested with avian influenza H5 subtype HI antigen, avian influenza H9 subtype Type HI antigen, Newcastle disease HI antigen and infectious bronchitis HI antigen were detected after 30-fold dilution with 0.85% sodium chloride solution. The results are shown in Table 7. The kit of the present invention has a longer storage period at 4° C., and the storage period is at least 90 days.

The shelf life test results under different storage conditions of the test kit in table 7

Note: "+" indicates that the detection line develops color, and "-" indicates that the detection line does not develop color.

Although the content of the present invention is described in conjunction with this embodiment, it should not be regarded as limiting the scope of the present invention, which is defined by the appended claims. In addition, those skilled in the art may make various changes or modifications to the present invention within the scope defined by the appended claims, and these changes or modifications also fall within the protection scope of the present invention.

Claims (4)

1. the hybridoma cell strain of the anti-avian infectious bronchitis virus monoclonal antibody of secretionXYCDY-1BV-3G5, is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCCNO:C201450.

2. the hybridoma cell strain of the anti-avian infectious bronchitis virus monoclonal antibody of secretionXYCDY-1BV-1E6, is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCCNO:C201454.

3. the hybridoma cell strain of the anti-avian infectious bronchitis virus monoclonal antibody of secretion claimed in claim 1The application of XYCDY-1BV-3G5 in the monoclonal antibody of preparation detection avian infectious bronchitis virus antigen.

4. the hybridoma cell strain of the anti-avian infectious bronchitis virus monoclonal antibody of secretion claimed in claim 2The application of XYCDY-1BV-1E6 in the monoclonal antibody of preparation detection avian infectious bronchitis virus antigen.