CN105602907A - Infectious bronchitis virus resistance monoclonal antibodies - Google Patents
Infectious bronchitis virus resistance monoclonal antibodies Download PDFInfo
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- CN105602907A CN105602907A CN201610051163.1A CN201610051163A CN105602907A CN 105602907 A CN105602907 A CN 105602907A CN 201610051163 A CN201610051163 A CN 201610051163A CN 105602907 A CN105602907 A CN 105602907A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
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Abstract
The invention belongs to the field of animal-borne disease immunodetection, and particularly relates to infectious bronchitis virus resistance monoclonal antibodies. One monoclonal antibody is secreted by a hybridoma cell strain XYCDY-1BV-3G5, wherein the cell strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:C201450. The other monoclonal antibody is secreted by a hybridoma cell strain XYCDY-1BV-1E6 of the infectious bronchitis virus resistance monoclonal antibody, wherein the cell strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:C201454. The hybridoma cell strains can be applied in preparation and detection of the monoclonal antibodies of the infectious bronchitis viral antigen.
Description
The present invention is that application number is the divisional application of 2014100921707 application cases, and the original bill applying date is on March 13rd, 2014.
Technical field
The invention belongs to animal immune applied technical field. Be specifically related to a kind of anti-avian infectious bronchitis virus monoclonal antibody,This monoclonal antibody can be at three kinds of breathing problems of fowl (bird flu H5 hypotype and H9 hypotype, ewcastle disease and avian infectious bronchusScorching) apply in three quick detection kit that detect of cause of disease. Kit prepared by the present invention can once complete three kinds of fowl simultaneouslyBreathing problem is as bird flu H5 hypotype and the former fast detecting of H9 hypotype, ewcastle disease and infectious bronchitis.
Background technology
The respiratory tract infection of fowl is the generation complexity of a class very common disease, especially such disease in avian production practiceChangeable, bring huge economic loss to aviculture. The cause of disease of respiratory tract infection that causes fowl mainly comprise virus, bacterium andMycoplasma, the three kinds of disease bird flus (Avianinfluenza, AI) that wherein cause by RNA virus, ewcastle disease (NewcastleDisease, ND) and infectious bronchitis of chicken (Infectiouslaryngotrache, IB) be comparatively common in chicken farm. Three kinds of diseasesSick have similar respiratory symptom clinically, and clinical manifestation mostly is atypical in addition, and disease infects and mostly is Combination, only certificateClinical symptoms and pathological manifestations are difficult to make a definite diagnosis, and therefore how making rapidly and accurately diagnosis is effective prophylactic treatment disease, avoids makingBecome serious economic loss basic.
At present, the method for bird flu, ewcastle disease and infectious bronchitis of chicken being made a definite diagnosis is mainly diagnosis of molecular biology method and bloodThe clear diagnosis of learning. Diagnosis of molecular biology technology mainly contains RT-PCR, Nucleic Acid Probe Technique, and Restriction fragment length is polymorphicProperty analysis etc. Serodiagnosis mainly contains agar gel diffusion test (AGID), hemagglutination test and hemagglutination-inhibition test (HA/HI),Immunofluorescence technique, ELISA (ELISA) and immunochromatographic method etc. Diagnosis of molecular biology method has high specificity, spiritSensitivity high, but experimental facilities, experimenter are had to higher requirement. Agar gel diffusion test in serodiagnosis method,Hemagglutination test and hemagglutination-inhibition test operation are comparatively simple, but its specificity and sensitivity are not high, and other are as immunofluorescence, and enzyme joinsImmunization is equally to equipment, and personnel and operation have high request. Said method is mostly consuming time long (as RT-PCR needs 1.5～2 hours;Agar gel diffusion test needs 24～48 hours; Hemagglutination-inhibition test needs 2～3 hours; ELISA test needs 3～5 hours etc.), noBe convenient in good time and quick diagnosis, so all cannot promote the use of in basic unit on a large scale.
Colloidal gold immunochromatographimethod (gold-immunochromatographyassayGICA) is using collaurum as tracer, based onColloidal gold-labeled method is applied to a kind of Novel immune labelling technique of antigen-antibody reaction. Its core technology is with celluloidFilm is solid phase carrier, because capillarity is impelled sample solution swimming on chromatography strip, and makes thing to be checked and chromatography in sampleOn film, there is at short notice the immune response of high specific, high-affinity for the acceptor (as antigen or antibody) of thing to be checked. ItHave easy, fast, high specificity, highly sensitive, the advantage that expense is low.
Based on this technology, cure disease surveillance people both at home and abroad, environmental hazard monitoring, food security guarantee and livestock and poultry detection etc.Field, has all developed multiple colloidal gold immunochromatographimethod fast detecting test card. Chicken disease diagnosis and context of detection, chickenEwcastle disease, infectious bronchitis of chicken, infections chicken cloacal bursa and mycoplasma Gallisepticum immune body immune colloidal gold quick detection kitObtain the Patents (patent No.: 200710169103.0; 200710169105.X; 200710169104.5; 200710169106.4).
But mentioned reagent box is the detection to antibody horizontal after relevant disease or vaccine immunity, and can accurately not react chicken group isNo infection epidemic disease. Although also have the immune colloid gold detection method of part for single cause of disease at present, as number of patent application is200510042631.0 " avian influenza virus antigen detection method and golden label fast diagnosis reagent box thereof and preparation method ", patent ShenPlease number be that 200510057023.7 document " bird flu immune colloid gold diagnosis test paper and test card " discloses several detection fowl streamThe preparation method of Influenza Virus antigen, but not good for the diagnosis effect of the case of mixed infection, use multiple single inspection collaurumsTest strips detects and has caused the waste of time and resource. And multivariate detection can disposablely be examined for multiple cause of diseasesSurvey, thereby well solved this problem. The combined detection kit (test card) of ternary can make consume be reduced to three/One, thereby for society saves ample resources, for the effective fast treating of disease provides the more sufficient time.
The present invention, by preparing respectively each two strains of bird flu, ewcastle disease and infectious bronchitis of chicken monoclonal antibody, utilizes dual anti-Whether sandwich immunoassay chromatographic technique, synchronously can detect in sample with single test card is bird flu, ewcastle disease and avian infectiousTracheitis cause of disease.
Summary of the invention
The object of the invention is to overcome the defect of existing detection technique, a kind of anti-avian infectious bronchitis virus monoclonal is providedAntibody, this monoclonal antibody can be at three kinds of breathing problems of fowl (bird flu H5 hypotype and H9 hypotype, ewcastle disease and avian infectiousBronchitis) cause of disease detect three quick detection kit in apply. Kit high specificity of the present invention, highly sensitive,Easy and simple to handle, can be used for synchronously completing bird flu H5 and H9 hypotype, ewcastle disease and three kinds of fowl of avian infectious bronchitis virus and exhaleInhale the detection of tract disease cause of disease.
The invention still further relates to structure and the exploitation of the hybridoma cell strain of six strain secretion monoclonal antibody specifics.
General technical route map of the present invention as shown in Figure 1.
The present invention is achieved through the following technical solutions:
In order to realize the present invention, applicant has prepared respectively two kinds for different antigen sites and can divide anti-avian influenza virus (to be called for shortAIV) hybridoma cell strain XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7, two kinds of secretion anti-new castle disease virus (are called for shortNDV) hybridoma cell strain XYCDY-NDV-3E11 and XYCDY-NDV-4B10 and two kinds of anti-avian infectious bronchuses of secretionScorching virus (being called for short IBV) hybridoma cell strain XYCDY-1BV-3G5 and XYCDY-1BV-1E6. Above-mentioned six strain cells all inDeliver China on March 11st, 2014. Wuhan. Wuhan University's Chinese Typical Representative culture collection center (CCTCC) preservation, corresponding guarantorTibetan information is summarized in as table 1.
The preservation information of six strain of hybridoma strains prepared by table 1 the present invention
On this basis applicant provide three kinds of breathing problems of fowl three fast fast bird flus (H5 and H9 hypotype),The kit of ewcastle disease and avian infectious bronchitis virus, this kit comprises test card and sample diluting liquid, wherein test cardBy sample pad (1), gold mark pad (2), nitrocellulose filter (3), absorption pad (4), PVC backing (9) and test get stuck (10)Composition, its concrete structure is: on PVC backing (9), be stained with successively in order sample pad (1), gold mark pad (2), nitric acidCellulose membrane (3), absorption pad (4); Test strips is packed into and tests formation test card in get stuck (10). At described gold mark pad (2)On be coated with three kinds of described gold mark monoclonal antibody mixed solutions (this solution contain anti-avian influenza virus monoclonal antibodyXYCSJL-AIV-2B8-colloid gold label thing, anti-new castle disease virus monoclonal antibody XYCDY-NDV-3E11-collaurum markNote thing and anti-avian infectious bronchitis virus monoclonal antibody XYCDY-1BV-3G5-colloid gold label thing); Described nitric acidOn cellulose membrane (3), be coated with anti-avian influenza virus monoclonal antibody XYCSJL-AIV-4B7, anti-new castle disease virus monoclonal is anti-Body XYCDY-NDV-4B10 and anti-avian infectious bronchitis virus monoclonal antibody XYCDY-1BV-1E6 form respectivelyDetection line T1 (5), T2 (6) and T3 (7); Nature controlling line (8) contains rabbit anti-mouse igg.
Applicant provides a kind of preparation to be fast applicable to bird flu H5 hypotype and H9 hypotype, ewcastle disease and infectious bronchitis of chickenThe method of three quick detection kit of virus causing disease, concrete steps are as follows:
1) avian influenza virus restructuring hemagglutinin HA1 albumen (being called for short AIV-HA1), newcastle disease virus restructuring hemagglutinin HN egg(be called for short NDV-HN) in vain and the expression preparation of avian infectious bronchitis virus recombinant nuclear capsid N albumen (being called for short IBV-NP). LogicalCross the sequence clone to bird flu HA1 gene, ewcastle disease HN gene and infectious bronchitis of chicken N gene, and connect correspondingThereby carrier builds three kinds of expression of recombinant proteins carriers pKG-HA1, pKG-HN and pKG-NP. Wherein expression vectorThe physical build-up collection of illustrative plates of pKG-HA1 is referring to document: middle National IP Network Dissertations Database: Wu Renwei, avian influenza virus recombinant protein is stickyFoundation [D] Hua Zhong Agriculture University of film immune Research and N-ELISA antibody detection method,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. And the physics structure of expression vector pKG-HN and pKG-NPBuild collection of illustrative plates as shown in Figures 4 and 5.
2) AIV-HA1, the purifying of NDV-HN and tri-kinds of albumen of IBV-NP. Recombinant protein purification chromatographic column used isGSTrapTMFFcolumns (buying from GEHealthcare.USA). Purification of recombinant proteins is pressed GSTrapTMFFcolumns saysBright book carries out.
3), with three kinds of recombinant protein A IV-HA1 after purifying, NDV-HN and IBV-NP respectively immune Balb/C mouse (purchaseFrom Wuhan Biological Products Inst.'s Experimental Animal Center), obtain anti-avian influenza virus (AIV) hybridoma cell strainXYCSJL-AIV-2B8 and XYCSJL-AIV-4B7, anti-new castle disease virus (NDV) hybridoma cell strainXYCDY-NDV-3E11 and XYCDY-NDV-4B10, anti-avian infectious bronchitis virus (IBV) hybridoma cell strainXYCDY-1BV-3G5 and XYCDY-1BV-1E6, the Balb/C mouse of hybridoma cell strain being injected to treated mistake, obtainsContaining the ascites of monoclonal antibody, through caprylic acid-ammonium, (Zhu Liping edits " immunology common experimental method ", People's Medical Officer Press, 2000Version) obtain the monoclonal antibody of purifying. Six strain cells have all been delivered China on March 11st, 2014. Wuhan. and China of Wuhan UniversityIn typical case's culture collection (CCTCC), preservation information is respectively: hybridoma cell strain XYCSJL-AIV-2B8, and its preserving number:CCTCCNO:C201449; Hybridoma cell strain XYCSJL-AIV-4B7, its preserving number: CCTCCNO:C201453;Hybridoma cell strain XYCDY-NDV-3E11, its preserving number: CCTCCNO:C201451; Hybridoma cell strainXYCDY-NDV-4B10, its preserving number: CCTCCNO:C201455; Hybridoma cell strain XYCDY-IBV-3G5,Its preserving number: CCTCCNO:C201450; Hybridoma cell strain XYCDY-IBV-1E6, its preserving number: CCTCCNO:C201454；
4) extract mouse IgG immune health new zealand rabbit (purchased from Wuhan Biological Products Inst.'s Experimental Animal Center), obtain rabbitDynamics;
5) react and prepare collaurum with gold chloride (purchased from sigma company) with trisodium citrate;
6) by step 3) the anti-AIV antibody (XYCSJL-AIV-2B8) prepared, anti-NDV antibody (XYCDY-NDV-3E11)Add respectively step 5 with anti-IBV antibody (XYCDY-1BV-3G5)) in the collaurum prepared, obtain three kinds of monoclonal antibodies-colloid gold label thing is pressed certain volume and is mixed the concentrated gold mark mixture that forms afterwards;
7) three kinds of gold mark mixtures are coated on gold mark pad (2);
8) by step 3) the anti-AIV antibody (XYCSJL-AIV-4B7) prepared, anti-NDV antibody (XYCDY-NDV-4B10)Be coated on successively and on nitrocellulose filter (3), form respectively detection line T1 (5) with anti-IBV antibody (XYCDY-1BV-1E6),T2 (6) and T3 (7); And rabbit anti-mouse igg is coated on to nitrocellulose filter (3) upper formation nature controlling line (8);
9) on described PVC backing (9), adhere to successively in order described sample pad (1), gold mark pad (2), nitric acid fibreTie up plain film (3), absorption pad (4), obtain described AIV (H5 or H9), the multivariate detection immunity colloid of NDV and IBVGold test paper strip.
10) by 9) described in test strips pack test into and get stuck and form test card in (10).
Obviously, the present invention prepare six secretion specific monoclonal antibodies hybridomas can be by allogenic disease seed culture of virusesClass is divided into three compositions to making, uses with forming double-antibody sandwich, wherein:
The hybridoma XYCSJL-AIV-2B8 of secretion anti-avian influenza virus monoclonal antibody and XYCSJL-AIV-4B7 are in systemIn standby detection avian influenza virus H5 and H9 hypotype antigen, it is paired use.
Hybridoma XYCDY-NDV-3E11 and the XYCDY-NDV-4B10 of secretion anti-new castle disease virus monoclonal antibodyDetecting in NDV antigen in preparation is paired use.
Secrete anti-avian infectious bronchitis virus monoclonal antibody hybridoma XYCDY-1BV-3G5 andXYCDY-1BV-1E6 is paired use detecting in NDV antigen.
Because this kit has adopted above-mentioned double antibody sandwich method coated antibody, thereby can greatly improve kit of the present inventionDetection effect.
Principle of the present invention is to adopt double antibody sandwich method (for the conventional method of report), with the coated anti-AIV antibody of detection line T1(XYCSJL-AIV-4B7) detect in sample whether contain avian influenza virus (AIVH5 or AIVH9) for example describes,In the time containing AIV (H5 or H9) in sample to be checked, the AIV (H5 or H9) in sample can be at gold mark pad position and gold markAnti-AIV antibody (XYCSJL-AIV-2B8) reacts, and while reacting compound chromatography to detection line position, is coated on detectionAIV (H5 or H9) in anti-AIV antibody meeting and compound on line T1 reacts, thereby forms in detection line position" golden labeling antibody-AIV-coated antibody " double-antibody sandwich compound is colloid gold label owing to there being a kind of antibody in compound, so just there will be macroscopic red stripes in detection line position, so just obtain positive findings. Have neither part nor lot in reactionGold labeling antibody chromatography is to nature controlling line position, and the lgG of the coated anti-mouse of rabbit catches and develops the color. On the contrary, if do not contained in sampleWhen AIV (H5 or H9), not containing double-antibody sandwich compound, just there will not be red stripes, so just in detection line positionObtain negative findings. Test strip (structure chart as shown in Figure 2) in kit of the present invention is by sample pad (1), gold mark pad(2), nitrocellulose filter (3), absorption pad (4) stick to successively and assemble on PVC backing (9) by illustrated order, test paperBar packs into tests formation test card in get stuck (10).
Compared with prior art, the present invention has advantages of following outstanding:
1, polynary fast detecting bird flu H5 of the present invention and H9 hypotype, ewcastle disease and avian infectious bronchitis virus collaurumKit, has high specificity, highly sensitive, and detection time, short (10-15 minute) judged the advantages such as directly perceived.
2, kit of the present invention is without any need for specific apparatus, equipment, and testing cost is low. Simultaneously because the present invention realizes threePlant the same of the main breathing problem of fowl (bird flu H5 and H9 hypotype, ewcastle disease and avian infectious bronchitis virus) cause of diseaseIn time, is detected, thereby has more effectively saved detection time and testing cost.
3, kit of the present invention is easy and simple to handle.
4, kit of the present invention stores conveniently, and less demanding to storage temperature, the shelf-life at 4 DEG C is at least 90 days.
Brief description of the drawings
Fig. 1: general technical route map of the present invention.
Fig. 2: the assembling schematic diagram of test strip of the present invention.
1-sample pad in figure, 2-gold mark pad, 3-nitrocellulose filter, 4-absorption pad, 5-detection line T1,6-detection line T2,7-detection line T3,8-nature controlling line, 9-PVC backing, 10-test is got stuck.
Fig. 3: the present invention examines test card result and judges schematic diagram.
In figure: 11: represent to detect AIV, NDV, IBV is positive findings; 12: represent to detect AIV positive, NDVBe negative findings with IBV; 13: represent to detect NDV positive, AIV and IBV are negative findings; 14: represent inspectionSurvey IBV positive, AIV and NDV are negative findings; 15: represent to detect AIV, NDV, IBV is weak positive knotReally; 16,17: represent that test card lost efficacy.
Fig. 4: the physical build-up figure that the present invention relates to the expression of recombinant proteins plasmid of NDV-HN.
Fig. 5: the physical build-up figure that the present invention relates to the expression of recombinant proteins plasmid of IBV-NP.
Detailed description of the invention
Embodiment 1
1.AIV-HA1 albumen, the preparation of NDV-HN albumen and IBV-NP albumen
AIV-HA1 albumen, the preparation method of NDV-HN albumen and IBV-NP albumen is mainly referring to document: Pehanorm Brooker J,Not Ritchie EF, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate). molecular cloning experiment guide. and the second edition, science goes outVersion society, the method providing in 1992 is carried out.
The structure of the clone of 1.1 HA 1 Gene of AIVs and order-checking and expression vector pKG-NS1
The construction method of the HA 1 Gene of AIV the present invention relates to and recombinant expression plasmid pKG-HA1 thereof and structure collection of illustrative platesKnow on the net and publish in China in 2006, referring to document: middle National IP Network Dissertations Database: Wu Renwei, avian influenza virus weightFoundation [D] Hua Zhong Agriculture University of the research of histone mucosal immunity and N-ELISA antibody detection method, 2006; Referring to network address: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；
The structure of the clone of 1.2 ewcastle disease HN genes and order-checking and expression vector pKG-HN
The sequence of the ewcastle disease HN gene the present invention relates to and the construction method of cloning process and expression vector pKG-HN are with reference to literary compositionOffer: middle National IP Network Dissertations Database: Sun Shuna, the clonal expression of F gene of NDV strain and HN gene and ELISA antibody thereofThe preliminary foundation [D] of detection method, Hua Zhong Agriculture University, 2006; Referring to network address: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。
Specific implementation method is as follows: a strain ewcastle disease NDVLa who preserves with Hua Zhong Agriculture University microorganism and the freeze-drying of immunization experiment chamberSota is strain in allantoic cavity inoculation NDV in healthy SPF (no-special pathogen) chicken of 9-11 age in days embryo (purchased from Wuhan biological productsResearch institute), abandon dead germ before 24 hours, collect the chick embryo allantoic liquid of 24-96 hour; Under 4 DEG C of conditions 4, the centrifugal 30min of 000rpm,Get supernatant; Under 4 DEG C of conditions 30, the centrifugal 60min concentrating virus of 000rpm. With PBS (DEPC the processes water preparation) dissolving of pH7.2Precipitation, after packing ,-70 DEG C save backup. The NDVLaSota including according to GenBank is the (login of separated strain HN gene orderNumber: AJ629060.1) design and synthesize primer, amplification HN gene. Upstream primer P1:ATAGGATCCGGGGCTAGCACACTTA; Downstream primer P2:AGCAAGCTTCTAGCCAGACTTGGCT. P1 is positioned at promoter upstream, is added with BamHI site, and P2 includes terminator codon, has added HindIII site, two enzymesCutting site underscore marks. It between two primers, is the HN gene of disappearance cytoplasmic region and cross-film region sequence. Getting appropriate viral suspension carriesGet RNA, by the method providing in TaKaRaRNAPCRKit (AMV) Ver.2.1 description, amplify DNA, adoptUNIQ-10 pillar DNA glue reclaims kit (E.Z.N.AGelExtractionKit) and reclaims purifying RT-PCR amplified production, willThe RT-PCR product reclaiming directly with carry pMD18-T purchased from the clone of TaKaRa company and carry out coupled reaction, then will connect productThing is transformed in the middle competent cell bacillus coli DH 5 alpha of kit (purchased from the commercial reagents box of TaKaRa company), picking sunSex clone, employing alkaline lysis (Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate) for Pehanorm Brooker J, not Ritchie EF. pointSub-cloning experimentation guide. the second edition, Beijing Science Press, 1992 editions) extract recombinant plasmid dna, and to recombinant plasmid dnaIdentify. By recombinant plasmid called after pMT-HN correct qualification, and serve Hai Shenggong bioengineering Co., Ltd and surveyOrder, then carries out the relevant data in sequencing result and GeneBank homology relatively and point folding.
PMT-HN cuts with BamHI and HindIII enzyme, reclaims the fragment of about 1590pb, then by this fragment and enzyme with sameThe expression vector pGEX-KG of the AmershamBiosciences company that enzyme is cut connects, construction of expression vector. Recombinant expression carrierTransform the DH5 α competent cell of fresh preparation, and transformed bacteria coating was cultivated containing 37 DEG C of the LB agar plates of ampicillinNight. After choosing several single bacterium colony overnight incubation, prepare plasmid in a small amount with alkaline lysis, carry out restriction analysis and pcr amplification qualification. WillThe positive colony called after pKG-HN obtaining. Positive colony is prepared to DNA in a large number with alkaline lysis, and packing frozen in-20 DEG C. Recombinant expression plasmid pKG-HN builds flow process as shown in Figure 4.
The structure of the clone of 1.3 infectious bronchitis of chicken N genes and order-checking and expression vector pKG-NP
The ewcastle disease the present invention relates to, the sequence of infectious bronchitis of chicken N gene and cloning process and expression vector pKG-NPConstruction method bibliography: middle National IP Network Dissertations Database: Li Zhonghua, IBV spike protein, core eggClone, expression and the Preliminary Applications in antibody test [D] thereof of white gene, Hua Zhong Agriculture University, 2006; Referring to network address: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. Specific implementation method is as follows: with Hua Zhong Agriculture University veterinary microorganism withStrain infectious bronchitis of chicken IBV (H52strain) strain that immunization experiment chamber freeze-drying is preserved in allantoic cavity inoculation NDV inHealthy SPF (no-special pathogen) the chicken embryo of 9-11 age in days (purchased from Wuhan Biological Products Inst.), abandons dead germ before 24 hours, receivesCollect the chick embryo allantoic liquid of 24-96 hour; Under 4 DEG C of conditions 4, the centrifugal 30min of 000rpm, gets supernatant; Under 4 DEG C of conditions 30,000rpmCentrifugal 60min concentrating virus. With PBS (DEPC the processes water preparation) dissolution precipitation of pH7.2, after packing ,-70 DEG C of preservations are standbyWith. The N gene order (number of logining: EF602459.1) of the IBV including according to GenBank is to comparatively conservative in its N geneSequences Design synthetic primer, amplification N gene. Upstream primer P1:GGATCCATGGCAAGCGGTAAAGCAG; Downstream primer P2:CCGAGCTCTCAAAGTTCATTCTCTCC. P1 is positioned at promoter upstream,Be added with BamHI point, P2 includes terminator codon, has added Sca I site, and two restriction enzyme sites mark with underscore. Two drawIt between thing, is fragment comparatively conservative in N gene. Get appropriate viral suspension and extract RNA, by TaKaRaRNAPCRKit(AMV) method providing in Ver.2.1 description, amplifies DNA, adopts UNIQ-10 pillar DNA glue to reclaim kit(E.Z.N.AGelExtractionKit) reclaim purifying RT-PCR amplified production, by the RT-PCR product of recovery directly with purchaseCarry pMD18-T from the clone of TaKaRa company and carry out coupled reaction, then connection product is transformed into kit (purchased fromThe commercial reagents box of TaKaRa company) in competent cell bacillus coli DH 5 alpha, picking positive colony, adopts alkaline lysis(Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate) for Pehanorm Brooker J, not Ritchie EF. molecular cloning experiment guide. theTwo editions, Beijing Science Press, 1992 editions) extract recombinant plasmid dna, and recombinant plasmid dna is identified. To reflectFixed correct recombinant plasmid called after pMT-N, and serve Hai Sheng work biotech firm and check order, then by sequencing result withRelevant data in GeneBank carries out homology relatively and point folding.
PMT-N cuts with BamHI and Sca I enzyme, reclaims the fragment of about 1.2kb, then by this fragment and enzyme enzyme with sameThe expression vector pGEX-KG of the AmershamBiosciences company of cutting connects, construction of expression vector. Recombinant expression carrierTransform the DH5 α competent cell of fresh preparation, and 37 DEG C of cultivations of LB agar plate containing ampicillin by transformed bacteria coatingSpend the night. After choosing several single bacterium colony overnight incubation, prepare plasmid in a small amount with alkaline lysis, carry out restriction analysis and pcr amplification qualification.By the positive colony called after pKG-NP obtaining. Positive colony is prepared to DNA in a large number with alkaline lysis, and packing is frozenIn-20 DEG C. Recombinant expression plasmid pKG-NP builds flow process as shown in Figure 5.
1.4AIV-HA1, the expression of NDV-HN and tri-kinds of albumen of IBV-NP
Transform respectively Escherichia coli with recombinant plasmid pKG-HA1, pKG-HN and pKG-NP and expression vector pGEX-KGBL21 (DE3), be coated with containing the LB flat board of 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), to put 37 DEG C of insulating boxs and cultivate, picking list bacterium colony, connectsPlant liquid LB culture medium (1% (W/V) Tryptone containing ampicillin (Amp) in 2.0mL; 0.5% (W/V) YeastExtract; 0.5% (W/V) NaCl and 0.1mg/mLAmp) in, 37 DEG C of 250-300r/min shaken cultivation, work as OD600nmWhile reaching 0.6-1.0. Inoculate respectively 1.0mL bacterium liquid and contain in the liquid LB culture medium of ampicillin (Amp) to 10.0mL,37 DEG C of 250-300r/min shaken cultivation 3h, then adding final concentration is derivant IPTG (isopropyl-β-D-of 0.024mg/mLSulfo-galactopyranoside), to induce 4 hours, centrifugal collection thalline carries out SDS-PAGE detection.
1.5AIV-HA1, the purifying of NDV-HN and tri-kinds of albumen of IBV-NP
(1) purifying of supernatant
According to a large amount of abduction deliverings of condition in above-mentioned 1.4, by the bacterial cultures after induction in the centrifugal 10min of 8,000r/min,PBS (phosphate) buffer solution (140mMNaCl, 2.4mMKCl, 10mMNa2HPO4.12H2O, 1.8mM for precipitationKH2PO4pH7.4) (be about the l/10 of original bacteria liquid volume) resuspended, add DTT (dithiothreitol (DTT)) to final concentration be 10mmol/L,With pressure breaking instrument be crushed to liquid become transparent, 4 DEG C, the centrifugal 15min of 12,000r/min, broken with the membrane filtration of 0.22 μ mSupernatant after broken, with GSTrapTMFFcolumns chromatographic column through AKTA protein purification system AKTAexplorer10 (purchased fromGEHealthcare company, USA) above-mentioned supernatant is carried out to purifying.
(2) purifying of inclusion body and renaturation
PBS for precipitation (pH7.4) after centrifugal fragmentation in above-mentioned (1) is washed 2 times, add 19.7mL buffer A (50mMTris-Cl, 0.5mMEDTA, 50mMNaCl, 5% (W/V) Glycerol) and 0.3mL20% (W/V) SKL (tenDialkyl group sodium sarcosinate) storage liquid, vigorous agitation, dissolves it, leaves standstill 2h, in 4 DEG C of centrifugal 10min of 12000r/min,Abandon precipitation, get supernatant, add 20% (W/V) PEG-4000 (Macrogol 4000) to final concentration be 0.2%, add 50mmol/LOxidized form glutathione to final concentration be 1mmol/L, reduced glutathione to the final concentration that adds 100mmol/L is 2Mmol/L, leaves standstill 2h, and with PBS (pH7.4) dialysis 2～3 days ,-70 DEG C saved backup.
(3) mensuration of protein concentration and qualification
With nucleic acid-protein analyzer measure respectively the absorbance value of protein solution under 280nm and 260nm wavelength be A280,A260. Calculate protein concentration according to formula, the protein solution that reaches requirement concentration is distributed into 100 μ L/ pipes, get 50 μ L and enterRow SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) qualification and Western-blot analyze, and all the other put-70 DEG CSave backup.
SDS-PAGE detects: get the thalline of above-mentioned collection, with the abundant resuspended thalline of 40.0 μ LddH2O, add isopyknic addingSample buffer solution (250mMTris-HCl, 10% (W/V) SDS, 0.5% (W/V) bromophenol blue, 50% (V/V) glycerine,5% (V/V) beta-mercaptoethanol), 3-5min is boiled in water-bath, and then ice bath is waited for application of sample electrophoresis. Press Pehanorm Brooker J, notRitchie EF, Manny A Disi T chief editor; Jin Dongyan, Li Mengfeng etc. translate, " molecular cloning experiment guide ", the second edition, 1992The described method of version is prepared separation gel and spacer gel, adds 1 × Tris-glycine electrophoretic buffer (25mMTris-base, 0.1%(W/V) SDS, 0.25MGlycine), get the sample point sample of processing, electrophoresis 2h, takes off gel, coomassie brilliant blue R250Dyeing liquor (0.1% (W/V) coomassie brilliant blue R250,25% (V/V) isopropyl alcohol, 10% (V/V) glacial acetic acid) dyeing 2h,Then use SDS-PAGE destainer (5% (V/V) ethanol, 10% (V/V) glacial acetic acid) decolouring, observe and take a picture. And pass throughThe foreign protein content that gel thin-layer scanning analysis is expressed.
Western-blot analyze: for analyze expressed fusion AIV-HA1, NDV-HN with GST label andThe immunologic competence of IBV-NP, carries out SDS-PAGE electrophoresis by method described above, and the gel after electrophoresis is not dyed,Directly be transferred on NC film charged albumen with transfer device, with 15V electricity transfer printing 1.5h. After transfer printing finishes, by NC film inOnce, then NC film is proceeded to can heated sealant in rinsing in TBST (150mMNaCl, 20mMTris-HCl, 0.05%Tween20)Polybag in, add confining liquid (containing 1% (W/V) bovine serum albumin(BSA) by 0.1mL-0.15mL/cm2 (NC membrane area)TBST), drain as far as possible after the bubble in bag, heat airtight sack, room temperature is shaken gently 1-2h or 4 DEG C and is spent the night. ThenAbandon confining liquid, film is taken out, film is proceeded in new polybag, add through TBST and dilute by the amount of 0.1mL-0.15mL/cm2(1:150) the anti-AIV hyper-immune serum of chicken, the same eliminating bubble also seals sack, and room temperature effect 1h on shaking table washes with TBST6 times, each 5mim, proceeds to film in new plastic bag, adds through TBST dilution (1:5000) by 0.1mL-0.15mL/cm2The anti-chicken IgG-HRP of rabbit (purchased from GenScript company), room temperature reaction 1h, TBST washes 6 times, each 5min, more finallyWith TBS rinsing 2 times, add substrate solution (DAB-H2O2) colour developing with 0.01mol/LTris-Cl (pH7.6) preparation,Once there is protein band, use immediately ddH2O stops, i.e. observable photograph.
Calculate the target protein concentration of (protein concentration (mg/mL)=l.45 × A280-0.74 × A260) acquisition purifying divides according to formulaBe not: HA1--0.45mg/mL; HN--0.53mg/mL; N--0.90mg/mL. Utilize Western-blot analysis result to showThe AIV-HA1 albumen (62KDa) of the present invention restructuring, NDV-HN albumen (84KDa), IBV-NP albumen (72 and 60KDa)There is specific reaction with corresponding positive serum respectively, confirmed that these several expression products all have good antigenicity.
2. anti-AIV antibody, the preparation of anti-NDV antibody and anti-IBV antibody:
Utilize three kinds of prepared recombinant protein A IV-HA1 of applicant, NDV-HN and IBV-NP be immune Balb/C mouse respectively(purchased from Wuhan Biological Products Inst.'s Experimental Animal Center), immune programme for children is to get the solution of protein content 100 μ g and isopyknicFreund's complete adjuvant (purchased from sigma company) emulsification, injection mouse peritoneal, booster immunization after 21 days, uses Fu Shi instead incompleteAdjuvant (purchased from sigma company) emulsification, finally in merging first three day, (best and immunity last time is separated by more than 4 weeks), abdominal cavity is strongChange immunity, antigen amount doubles that (200 μ g), do not add adjuvant. When fusion, one of the Balb/C mouse of the last reinforced immunological of learning from else's experience,Eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked 5min sterilization in 75% alcohol. Aseptic taking-up mouse spleen,Isolate splenocyte, (SP2/0 myeloma cell is by Wuhan Biological Products Inst., Ministry of Public Health with the SP2/0 myeloma cell who recoversBe so kind as to give) mix in 50mL centrifuge tube in the ratio of 1～2 × 107 SP2/0 and 108 immunocytes (1:10～1:15),1500rpm, centrifugal 10min. Evacuation supernatant (filter paper of available sterilizing blots), knocks the pipe end gently, makes cell precipitation pine slightlyMoving. The centrifuge tube that cell mixture is housed is put in 37 DEG C of water-baths. Then in 1min, slowly splash into 50% of pre-temperature to 37 DEG CPEG0.8mL (purchased from sigma company), limit edged stirs with pipette tip gently, continues to stir 1min. Then slowly addThe 10mLRPMI-1640 basic culture solution (purchased from GIBCO company) of 37 DEG C of pre-temperature. Concrete grammar is: first minute dropwiseSplash into 1mL, within second minute, add 1ml, within 3rd～4 minutes, add 3mL, within the 5th minute, add remaining 5mL, each added-time needsSlowly add, and constantly stir lightly. Finally add 30mL1640 liquid, also need slowly to add. The centrifugal 5min of 800rpm,Remove supernatant, place 5～8min in 37 DEG C. With the suspension of HAT (purchased from GIBCO company) culture medium, also cultivate with HAT simultaneouslyBase suspend the raising splenocyte for preparing and with merge after mixing with cells, add as required appropriate HAT culture medium and (cultivateThe RPMI-1640 basic culture solution of the composition of base: 80mL, 20mL sterilizing calf serum, the HAT of 1mL100%, 1mL10, the mycillin of 000U/mL is dual anti-), point kind in 96 well culture plates, approximately 250 μ L/ holes. Single cell fusion can inoculate 4～8 96 orifice plates. Also can plant less as required, generally press SP2/0 myeloma cell's cell number and calculate, every hole inoculum concentration approximately containsA 104 left and right SP2/0 myeloma cell. In 37 DEG C, in 5%CO2 incubator, cultivate. Merge within latter second day, to start to observe and have or notPollute, added 1 HAT culture medium in the 4th day, within 8th～10 days, suck 100 μ L culture mediums and change HT (purchased from GIBCOCompany) culture medium 100 μ L. Treat that fused cell colony grows to culture hole 1/4, when culture medium omits flavescence, carry out antibody test. RightMerge hole in AI and use AIV-HA1 fusion and AIV allantoic fluid respectively as coating antigen, ND merges hole coating antigen and isNDV-HN fusion and NDV allantoic fluid, it is that IBV-NP merges and IBV allantoic fluid that IB merges hole coating antigen, all uses twoScreening detects, and utilizes conventional ELISA method to filter out the positive hole of secretion corresponding monoclonal antibody. To the positive screeningHole use at once limiting dilution assay (with reference to Xue Qingshan, " philosophy and technique of in vitro culture ", Science Press, calendar year 2001 version) enterRow clone, screening. Through 3～4 time clonings, finishing screen is selected secretion and is obtained anti-avian influenza virus (AIV) antibody hybridoma cellXYCSJL-AIV-2B8 and XYCSJL-AIV-4B7, anti-new castle disease virus (NDV) antibody hybridoma cellXYCDY-NDV-3E11 and XYCDY-NDV-4B10 and anti-avian infectious bronchitis virus (IBV) antibody hybridoma cellXYCDY-1BV-3G5 and XYCDY-1BV-1E6 totally 6 strain cells (biological preservation information is referring to " in invention of this descriptionHold " part). Above-mentioned 6 strain cells are carried out to chromosome counting, result demonstration, SP2/0 myeloma cell's is chromosomal averageNumber is 70, and splenocyte chromosome number is 40, and the chromosome number of hybridoma is between 80～94. All higher than twoThe chromosome number of parental cell, illustrates the SP2/0 myeloma cell really of fused cell and the hybridization product of splenocyte, hybridizationThe chromosome number of oncocyte is obviously more than SP2/0 myeloma cell's chromosome. With being added ELISA qualification anti-same cause of diseaseTwo strain clones are all the antibody producing for different epitopes. 6 strain cells of the present invention (1 × 106) are injected respectivelyBalb/C mouse peritoneal, manufacture order clonal antibody. Adopt the mouse source monoclonal antibody hypotype identification kit (Mouse of ROCKLAND companyMabIsotypingTestKit) monoclonal antibody the present invention being obtained is carried out hypotype qualification, and result is mouse IgG 2b AsiaClass.
3. the purifying of monoclonal antibody
With reference to Zhu Li equality, " immunology common experimental method ", People's Medical Officer Press, the method for report in 2000 editions: getThe mouse ascites 5mL of gained mixes with appropriate silica, adds isopyknic barbitol buffer solution (formula: sodium chloride85.00g, barbital 5.75g, barbital sodium 3.75g, Sodium azide 2.00g, is settled to 2000mL with distilled water), room temperature vibrationAfter 1h, at room temperature leave standstill 30min, get supernatant in clean centrifuge tube, in 4 DEG C, the centrifugal 10min of 3000rpm; GetClear liquid 8mL, adds 16mL0.06mol sodium-acetate buffer, with HCl adjust pH to 4.5, under fully stirring, slowly addsAfter sad 132 μ L, stirring at room temperature 30min, then proceeds to 4 DEG C of refrigerators and fully precipitates 2h, and 4 DEG C, the centrifugal 30min of 15000rpm,Obtain supernatant 22mL, add the phosphate buffer of 2.2mL0.1M (to be called for short PB, formula: 10mMNa2HPO4.12H2O and1.8mMKH2PO4PH7.2),, with NaOH adjust pH to 7.6, under stirring, slowly add ammonium sulfate to final concentration to be0.277g/mL, 4 DEG C of refrigerators fully precipitate after 2h, and in 4 DEG C, the centrifugal 30min of 12000rpm, abandons supernatant, precipitation 5mL0.01MPBS buffer solution (formula: 140mMNaCl, 2.4mMKCl, 10mMNa2HPO4.12H2O，1.8mMKH2PO4PH7.2) resuspended, pack bag filter into, after 5000mL0.01MpH7.2PBS buffer solution is fully dialysed, then to 2000mLDistill water dialysis, finally boils off ionized water dialysis to 3000mL tri-, and the protein solution of fully dialysing is dense with PEG-20000Be reduced to 3mL, then in 4 DEG C, the centrifugal 30min of 12000rpm, abandons precipitation, collects supernatant, records six strain monoclonal antibody concentration and exists1.0-2.2mg/mL between. Be accredited as the monoclonal antibody of purifying through SDS-PAGE, its purity is greater than 95%. This monoclonal is anti-Body can be used for preparing immune colloid gold.
4. the preparation of rabbit anti-mouse igg antibody:
Utilize Balb/C mouse IgG immune health new zealand white rabbit, high the exempting from of rabbit anti-mouse igg of preparing high specific, high-titerSerum, to hyper-immune serum adopt saturated ammonium sulphate method (bibliography: Zhu Li equality, " immunology common experimental method ",People's Medical Officer Press, 2000 editions) slightly carry, after crossing post, G-200 obtains highly purified rabbit anti-mouse igg antibody. ITo utilize this antibody be the core reagent as the nature controlling line of kit.
5. the preparation of monoclonal antibody-colloid gold label thing
(1) preparation of collaurum:
1% gold chloride is diluted to 0.01% with two ionized waters that boil off, puts on magnetic force heating stirrer and stir and boil, by every 100mL0.01% gold chloride adds 1.5mL1% trisodium citrate, continues to boil, until liquid is the orange red heating that stops, being cooled toAfter room temperature, supply dehydration. The collaurum outward appearance preparing should be pure, bright, without precipitation and floating thing, put 4 DEG C of preservations.
(2) preparation of monoclonal antibody-colloid gold label thing:
By the anti-AIV antibody XYCSJL-AIV-2B8 of gained, anti-NDV antibody XYCDY-NDV-3E11 and anti-IBV are anti-Body XYCDY-1BV-3G5 is mark colloid gold particle respectively. Concrete steps are as follows: under magnetic agitation, use 0.1M potashSolution is adjusted the pH value to 8.0 of collaurum, adds above-mentioned three kinds of monoclonal antibody (anti-AIV by 5～7.2 μ g antibody/mL collaurumsAntibody XYCSJL-AIV-2B8, anti-NDV antibody XYCDY-NDV-3E11 and anti-IBV antibody XYCDY-1BV-3G5),Continue to stir and evenly mix 30min, adding 10%BSA (bovine serum albumin(BSA)) is 1% to final concentration, standing 30min. 12000Rpm, 4 DEG C of centrifugal 30min, abandon supernatant, the borate buffer solution of 0.02MpH9.0 for precipitation (formula: boric acid 0.1237g,PEG-200001g, is settled to 1000mL with tri-distilled water, adjusts pH to 9.0) washed twice, with 1/20th initial colloidBorate buffer solution (the formula: boric acid 0.1237g, PEG-200001g, uses tri-distilled water constant volume of the 0.02MpH9.0 of gold volumeTo 1000mL, adjust pH to 9.0) will precipitate resuspended, put 4 DEG C for subsequent use, 60 days shelf-lifves.
6. gold mark pad is coated
Gold mark pad is soaked in to confining liquid (formula: 2%BSA, 2.5% sucrose, 0.3%PVPK-30,0.02%NaN3,0.5%Teewn-20，0.5％PEG-2000，10mMNa2HPO4.12H2O and 1.8mMKH2PO4PH7.2) after middle 30min,In 37 DEG C of oven dry. Get three kinds of monoclonal antibodies (anti-AIV antibody XYCSJL-AIV-2B8, the anti-NDV of isopyknic above-mentioned preparationAntibody XYCDY-NDV-3E11 and anti-IBV antibody XYCDY-1BV-3G5) antibody gold label thing fully mix rear concentrated,With Biodot point film instrument, the monoclonal antibody-colloid gold label thing preparing is evenly coated on to gold mark pad upper, every centimetre of gold mark padCoated 9 μ L antibody-colloid gold label things, vacuum drying (according to a conventional method), Vacuum Package (according to a conventional method), puts 4 DEG CFor subsequent use.
7. the processing of sample pad
Sample pad is soaked in to confining liquid and (contains 2%BSA, 2.5% sucrose, 0.3%PVPK-30,0.02%NaN3,0.5%Teewn-20，0.5％PEG-2000，10mMNa2HPO4.12H2O and 1.8mMKH2PO4PH6.4) after middle 30min,In 37 DEG C of oven dry, Vacuum Package, put 4 DEG C for subsequent use.
8. nitrocellulose filter is coated
To resist AIV antibody XYCSJL-with coating buffer (containing 3% methyl alcohol, the 0.01MpH7.4PBS buffer solution of 1% sucrose)AIV-4B7, anti-NDV antibody XYCDY-NDV-4B10 and anti-IBV antibody XYCDY-1BV-1E6 dilution 10-18 μ g/mL,It is coated on nitrocellulose filter successively as detection line (detection line is followed successively by T1, T2 and T3) with Biodot point film instrument,Package amount is 0.7 μ L/cm, and this detection line is near gold mark pad end, apart from the about 5mm of gold mark pad pad end; With coating buffer by rabbit anti-mouse iggAntibody dilution is to 500 μ g/mL, is coated in nitrocellulose filter as nature controlling line with Biodot point film instrument, and package amount is0.7 μ L/cm, this nature controlling line is near absorption pad, and apart from the about 5mm of absorption pad, nature controlling line or detection line between any two distance are 3～4mm.Dry 30-40min for 37 DEG C, for subsequent use.
9. the assembling of kit
Sample pad (1), gold mark pad (2), cellulose nitrate rope film (3), absorption pad (4) are sticked to PVC successively by the order shown in Fig. 2Backing (7) is upper, is cut into wide little of 4mm, is packaged in the test composition test card in (10) that gets stuck, after having prepared by this examinationTest card Vacuum Package in agent box. In 4 DEG C of preservations, the shelf-life is at least 90 days.
Embodiment 2 (Application Example)
The using method of main breathing problem three quick detection kit of fowl
1. the composition of kit, comprising:
1. 25 of test cards
2. one bottle of sample diluting liquid (10mL/ bottle)
2. the preparation of sample
2.1 sample diluting liquids: sample diluting liquid is 0.85% sodium chloride solution. Compound method: 8.5gNaCl, adding distil water is settled to1000mL。
2.2 sample preparation
The detailed method of operation of sample collection and processing is as follows:
(1) the pharyngeal sample of tracheae: first with hand, the howl of chicken is pushed aside, then with hand or tweezers by the tongue pull-out of chicken, expose pharyngeal,Cotton swab is inserted in pharyngeal tracheae, stirs several lower taking-ups, then put into that in advance oneself adds the sample cell of 500 μ L sample diluting liquids,Firmly stir, push, make as far as possible the sample on cotton swab elute, leave standstill after 15min, liquid in pipe supernatant is to be treatedSample product;
(2) the excrement stain sample on cloaca sample or fortune fowl vehicle: directly sample or dip fortune fowl vehicle with cotton swab from the cloaca of chickenOn excrement stain, then cotton swab is put into oneself adds the sample cell of 500 μ L sample diluting liquids in advance, the same elution samples, abandonsRemove cotton swab, the supernatant after leaving standstill in pipe is sample to be checked;
(3) muscle or internal organ sample: get chest muscle or thigh place muscle one fritter or internal organs one fritter (l-2g), add in millAfter entering 1-3mL sample diluting liquid and fully grinding, freeze thawing once, 4,000r/min, centrifugal 5min, supernatant is sample to be checked.
3. detect: take out kit, equilibrium at room temperature 20 minutes; Open the package, take out test card, get 100-120 μ L preparationGood sample splashes in the well of test card, result of determination in 10～15 minutes.
4. result is judged: as shown in Figure 4, the detection line T1 on NC film, T2 and T3 be corresponding AIVH5 or the AIVH9 of detecting respectively,NDV and IBV. When macroscopic aubergine appears in the nature controlling line on NC film in test card, and corresponding detection line is as T1There is not macroscopic aubergine in (or T2 or T3), detects in sample not containing AIVH5 or AIVH9 (or NDVOr IBV), result is judged to feminine gender, is designated as "-"; When macroscopic aubergine nature controlling line appears in test card, simultaneously correspondingDetection line there is macroscopic aubergine as T1 (or T2 or T3), detect in sample containing AIVH5 or AIVH9 (NDVOr IBV), result is judged to the positive, is designated as "+"; Virus quantity in detection line color depth and tested serum is proportionate, faceLook illustrates that the viral level of detected sample is higher more deeply, and nature controlling line occurs being judged to detection test card without band and lost efficacy.
Embodiment 3
The applicating example of main breathing problem three quick detection kit of 1 fowl
The detection of 1.1 chicken standard antigens
1. specific test: respectively by the bird flu H5 hypotype HI antigen being purchased, bird flu H9 hypotype HI antigen, chicken new city(above-mentioned biomaterial is studied purchased from the Harbin animal doctor of the Chinese Academy of Agricultural Sciences for epidemic disease HI antigen, infective bronchitis HI antigenInstitute), egg drop syndrome (EDSV-76) HI antigen, infections chicken cloacal bursa (IBD) agp antigen, chicken poison Mycoplasma antigen,Infectious laryngotracheitis of chicken (AILT) viral antigen (above-mentioned biomaterial is purchased from Beijing. China Veterinery Drug Inspection Office), 0.85% sodium chloride solution and negative allantoic fluid are tested by method (GICA) described in the embodiment of the present invention 2. In kit of the present inventionDetection test card detect bird flu H5 hypotype HI antigen, bird flu H9 hypotype HI antigen, newcastle disease HI antigen,When infective bronchitis HI antigen, all there is obvious aubergine band, the inspection of non-correspondence in nature controlling line and corresponding detection lineSurvey line not displaing amaranth band (as detected bird flu H5 hypotype HI antigen time, nature controlling line and detection line T1 occur simultaneouslyAubergine band, and there is not band in detection line T2 and T3; And detect 3 kinds of above-mentioned antigens simultaneously, be nature controlling line and detectionLine T1, there is aubergine band in T2 and T3 simultaneously); And detection 0.85% sodium chloride solution (blank), negative allantoic fluid,Egg drop syndrome (EDSV-76) HI antigen, infections chicken cloacal bursa (IBD) agp antigen, chicken poison Mycoplasma antigen and chicken passMetachromia laryngotracheitis virus antigen, there is aubergine band in nature controlling line only. This shows that the test card in kit of the present invention is specialProperty is high, shows with other important diseases antigen of fowl without any cross reaction.
Table 2 is applied test card of the present invention to fowl standard antigen specific detection result
Note: the colour developing of "+" detection line, "-" represents that detection line does not develop the color.
2. sensitivity tests: with 0.85% sodium chloride solution to bird flu H5 (H9) hypotype HI antigen, newcastle disease HI antigen,Doubling dilution after infective bronchitis HI antigen first dilutes 10 times respectively, final dilution factor is 1: 10240, respectively getsThe sample that 100-120 μ L has diluted is tested by kit test card of the present invention. The results are shown in Table 3. When bird flu H5 (orH9) when hypotype HI antigen doubling dilution is to 1:2560, newcastle disease HI antigen doubling dilution is during to 1:1280, and infectiousness is propped upTracheitis HI antigen doubling dilution to 1:640 test card is still positive, and shows that the test card sensitivity in this kit is higher.Prepare AIV, the test card that the individual event of NDV and tri-kinds of cause of diseases of IBV detects is tested its sensitiveness simultaneously, and result shows the present inventionSingle inspection test card the indifference of the test card in kit and three kinds of cause of diseases.
Table 3 test card of the present invention detects the sensitivity tests result of three kinds of standard antigens
The detection of 1.2 kits of the present invention to submitted sample and with the comparison of viral method for separating and detecting
With kit of the present invention, periphery chicken farm, Wuhan City, Hubei Province being gathered to 118 parts of tracheae swab samples altogether detects(preparation of sample is referring to embodiment 2), carries out virus to sample simultaneously and separates and identify, suppresses with blood clotting (HA) and blood clotting(HI) detect as contrast, wherein detect AIV referring to National Standard of the People's Republic of China GB/T18936-2003, detect NDVReferring to National Standard of the People's Republic of China GB/T16550-2008, detect IBV referring to National Standard of the People's Republic of ChinaGB/T23197-2008. The testing result contrasting between the two is in Table 4-6, as can be seen from Table 4, when detect AIVH5 (orH9) when hypotype, positive coincidence rate 70% and negative match-rate 90.7% that kit of the present invention and HA and HI detect, always symbolThe rate of closing is 92.3%. As can be seen from Table 5, in the time detecting NDV, the sun that kit of the present invention and HA and HI detectProperty coincidence rate 76.5% and negative match-rate 91.7%, total coincidence rate is 96.2%. And by seeing in table 6, when detecting IBVTime, positive coincidence rate 70.6% and negative match-rate 95.3% that kit of the present invention and HA and HI detect, total coincidence rate is95.7%. Result shows that the testing result of kit of the present invention and HA and HI detection method is basically identical, and coincidence rate is better.
Table 4 kit of the present invention and blood clotting (HA) and blood clotting suppress the Clinical detection AIV result that (HI) detects
Table 5 kit of the present invention and blood clotting (HA) and blood clotting suppress the Clinical detection NDV result that (HI) detects
Table 6 kit of the present invention and blood clotting (HA) and blood clotting suppress the Clinical detection IBV result that (HI) detects
Embodiment 4
The examination of three kinds of breathing problem three quick detection kit stability of fowl of the present invention
By the kit test card of the present invention in the Vacuum Package of 4 DEG C and 37 DEG C placements respectively, the the the 7th, 16,35,54,70,Within 90 days, take out, with bird flu H5 hypotype HI antigen, bird flu H9 hypotype HI antigen, newcastle disease HI antigen and infectiousness bronchusScorching HI antigen is with detecting after 30 times of dilutions of 0.85% sodium chloride solution. Result is as table 7, and kit of the present invention is the guarantor of 4 DEG CThe phase of depositing is longer, and storage life is at least 90 days.
Storage life result of the test under the different storage requirement of table 7 kit
Note: the colour developing of "+" detection line, "-" represents that detection line does not develop the color.
Although content of the present invention is to describe in conjunction with the present embodiment, can not think limitation of the scope of the invention, thisScope of invention is limited by appended claims. In addition, the scope that those skilled in the art limits at appended claimsIn the present invention is carried out to various changes or modification, these changes or modified forms drop in protection scope of the present invention equally.
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.
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| CN107840884A (en) * | 2017-11-15 | 2018-03-27 | 郑州大学 | A kind of nano antibody of anti-birds infectious bronchitis virus and preparation method thereof |
| CN111077318A (en) * | 2019-12-31 | 2020-04-28 | 贵州省烟草科学研究院 | Rapid test card for simultaneously detecting TMV, PVY and TVBMV and preparation and use methods thereof |
| CN113063939A (en) * | 2021-03-05 | 2021-07-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit and detection method for infectious bronchitis viruses and application |
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| CN110452885B (en) * | 2019-09-03 | 2022-11-04 | 江苏省农业科学院 | Hybridoma cell 4F6 strain secreting monoclonal antibody against newcastle disease virus NP protein |
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| EP1300157B1 (en) * | 2001-10-04 | 2005-10-26 | Centrum voor Onderzoek in Diergeneeskunde en Agrochemie | Attenuated mutant newcastle disease virus strains for in ovo vaccination and their use |
| US20100009339A1 (en) * | 2006-05-18 | 2010-01-14 | Nikolay Vladimirovich Bovin | Detection method for influenza viruses |
| JP5300200B2 (en) * | 2006-08-31 | 2013-09-25 | 大阪府 | Rapid diagnostic method specific to avian influenza virus |
| CN101376677A (en) * | 2008-09-25 | 2009-03-04 | 山东省农业科学院畜牧兽医研究所 | Monoclonal antibody IE5 for detecting new castle disease virus variation strain |
| KR101211593B1 (en) * | 2010-09-06 | 2013-01-07 | 유한회사 바이오노트 | Diagnostic kit for H9 type avian influenza virus using rapid immunochromatography and the method for diagnosing H9 type avian influenza by using the same |
| CN202693594U (en) * | 2012-07-11 | 2013-01-23 | 郑州大学 | Colloidal gold test strip for detecting H5-subtype avian influenza virus antibodies, Newcastle disease virus antibodies and chicken bursal disease virus antibodies |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107840884A (en) * | 2017-11-15 | 2018-03-27 | 郑州大学 | A kind of nano antibody of anti-birds infectious bronchitis virus and preparation method thereof |
| CN111077318A (en) * | 2019-12-31 | 2020-04-28 | 贵州省烟草科学研究院 | Rapid test card for simultaneously detecting TMV, PVY and TVBMV and preparation and use methods thereof |
| CN113063939A (en) * | 2021-03-05 | 2021-07-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit and detection method for infectious bronchitis viruses and application |
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| CN104007261B (en) | 2016-04-06 |
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