CN104330560B - Double sandwich-ELISA detection avian reticuloendotheliosis virus antigen test kit - Google Patents

Double sandwich-ELISA detection avian reticuloendotheliosis virus antigen test kit Download PDF

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CN104330560B
CN104330560B CN201410629638.1A CN201410629638A CN104330560B CN 104330560 B CN104330560 B CN 104330560B CN 201410629638 A CN201410629638 A CN 201410629638A CN 104330560 B CN104330560 B CN 104330560B
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avian reticuloendotheliosis
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CN104330560A (en
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秦爱建
邵红霞
钱琨
鲍衍清
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Yangzhou University
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • 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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Abstract

The test kit of double sandwich-ELISA detection avian reticuloendotheliosis virus (REV) antigen, belongs to field of biological technology detection, and this test kit, for the detection of REV P30 proteantigen, minimum can detect 101.82P30 antigen entrained by the REV HA1101 of individual TCID50 or SNV Strain.The quickly detection that the present invention is clinical REV antigen provides reliable means.This test kit is easy and simple to handle, quick, it is possible to for the detection of REV, it is adaptable to the REV rapid, high volume detections such as basic unit's Veterinary office at different levels, inspection and quarantining for import/export, production of vaccine enterprise.Needed for the method with low cost, remarkable in economical benefits, application prospect are extensive.

Description

Double sandwich-ELISA detection avian reticuloendotheliosis virus antigen test kit
Technical field
The invention belongs to field of biological technology detection, be specifically related to a kind of detection technique field to avian reticuloendotheliosis virus (REV) antigen.
Background technology
Avian reticuloendotheliosis is as a kind of oncogenicity and immunosuppressive disease, and its harm that poultry farming is caused is self-evident.But for various reasons, this disease does not obtain enough attention in many countries and regions.In recent years, avian reticuloendotheliosis virus (REV) infecting and the popular trend having increase poultry, and the production capacity of the often major injury fowl group of the mixed infection between other tumorigenic disease also causes certain economic loss, and poultry farming is adversely affected.
Since 1958, REV propagates already by number of ways in the fowl group of multiple countries and regions, presents worldwide distribution.Serologic detection according to carrying out in Korea S, Egypt, Taiwan and the U.S. in recent years shows, all there is REV antibody positive in multiple chicken groups and turkey group, and positive rate is from 34% to 92% not etc..At home, eighties of last century this disease eighties is only sporadic distribution in chicken group and duck group, in recent years, along with more carrying out about this disease Epidemiological study, finds there is higher REV antibody positive rate in a lot of chicken groups.PCR and Detection of antigen show, all there is REV in various degree and infect in broiler, laying hen, aquatic bird, and the recall rate of REV in the chicken group of China mainland area, is the highest in all detection samples.
The situation that REV and other several immunosuppressive diseases include MDV, ALV and CAV mixed infection is very general, is cause chicken group serious immunosuppressant occur, strengthen one of pathogenic major reason of other cause of diseases.Chicken group at ALV-J and REV coinfection, although the immunne response for REV does not have obvious suppression, but the immunne response for ALV-J then can seriously suppress, and the persistent period is 7 weeks, have a strong impact on the production capacity of chicken group, also hamper and work to ALV-J eradication, under experimental conditions, the mixed infection of REV and MDV can reduce body and produce the ability of IFN-γ, it is suppressed that body is for the natural immunity ability of virus.
In addition, the insertion in other viral genome of the REV viral nucleic acid is also one of emphasis that academia is paid close attention in recent years.In bird pox virus and Marek’s disease virus gene group, all occur part REV genome LTR sequence or only lack part 3 ' hold LTR big section of REV genome insert the genomic phenomenon of its viral DNA.In bird pox virus, compare with the bird pox virus being not inserted into REV fragment or Insert Fragment negligible amounts, insert REV genome a fairly large number of bird pox virus replication capacity in host and virulence is remarkably reinforced.And in the Marek’s disease poison inserting REV genetic fragment, after deleting the LTR fragment of REV, its growth and immunosuppression capability strengthen to some extent, but the horizontal transmission ability of MDV but substantially weakens, prompting REV fragment to be inserted in the communication process of MDV significant.
The separation of cause of disease and qualification are REV detection methods the most classical, the most reliable, generally use the internal organs lapping liquid of birds to be checked, whole blood or blood plasma inoculation permissive cell, re-use the maintenance medium containing 1% calf serum, blind passage 2 ~ 3 generation on permissive cell, per generation about week age, then re-uses the monoclonal antibody for REV and carries out immunofluorescence, SABC, complement fixation test etc. virus that may be present in cell culture is detected.This process length consuming time, and sensitivity, simplicity are also strong, but reliable results, often as the goldstandard of other detection methods with for referencial use.Sandwich ELISA or antibody capture ELISA method based on monoclonal antibody or polyvalent antibody can at short notice directly to REVenv or p in the suspicious sample that include anus swab and Ovum Gallus domesticus album in high volume30Antigenic component detects, and without carrying out virus amplification in advance on permissive cell, simple and effective, economy are convenient, have significantly high use value.But under experimental conditions, the ELISA detection method based on multi-resistance still suffers from higher false positive rate, monoclonal antibody is adopted to can solve the problem that this problem.
Serologic detection, can adopt multiple method that REV antibody or antigen in Suspect serum, Ovum Gallus domesticus album or yolk are detected, but the frequency of its appearance and persistent period inconsistent.Can be antigen with the fixing permissive cell infecting REV, Suspect serum sample is carried out IFA detection, it is also possible to virus neutralization experiment, agar gel precipitation experiment (AGP) or with gp90For the indirect ELISA of antigen or block ELISA experiment etc. and detect, wherein agar gel precipitation experiment can be used for the detection of antigen and antibody but susceptiveness is not high.
Therefore be presently required exploitation efficiently, quick, convenient, Financial cost is relatively low and can be used in the diagnostic method of extensive field sample detection.
Summary of the invention
The present invention seeks to set up the test kit of a kind of double sandwich-ELISA detection avian reticuloendotheliosis virus antigen, to adapt to diagnosis needs efficient, quick, convenient, that Financial cost is relatively low.
The present invention includes capture antibodies REVp30-2C8 and enzyme labelled antibody REVp30-5G6。
Identified, these monoclonal antibodies all can react with the P30 in the CEF pyrolysis product that REVHA1101 strain and SNV strain are infected, and does not react with the CEF product of cell lysis of the viral infection such as normal CEF cell, ALV-J, CAV, MDV or HVT.The test kit that the present invention sets up, adopts that ELISA method is minimum can detect 101.82Individual TCID50The P30 antigen entrained by REVHA1101 or SNV.Adopt this test kit, detection can be made to realize efficient, quick, convenient, economical, and can be used in the detection of avian reticuloendotheliosis virus (REV) in the sample of extensive field.
The present invention another object is that the application proposing double sandwich-ELISA detection avian reticuloendotheliosis virus (REV) antigenic reagent box.
The present invention adopts ELISA detection method, it is characterized as being: cell culture to be checked, cloaca swab or Ovum Gallus domesticus album are made, and the REVP30 recombinant products equal samples of vivoexpression, suitably after dilution, add in this test kit ELISA Plate, it is possible to whether detection sample contains the P30 antigen of REV or REV.
Adopt above method, whether judgement sample can contain the P30 antigen of avian reticuloendotheliosis viral (REV) or REV fast and efficiently.
Accompanying drawing explanation
Fig. 1 is the gel electrophoresis analysis figure of pcr amplification REVHA1101 strain p30 gene.
Fig. 2 is the abduction delivering analysis chart of recombiant protein.
Fig. 3 is REVp30-2C8 and REVp30The Monoclonal Antibody Cell supernatant IFA result picture of-5G6.
Fig. 4 is virus titer and the Detection results graph of a relation of test kit detection REVHA1101 or SNV.
Fig. 5 is the reaction result figure of test kit detection different virus or vivoexpression product.
Detailed description of the invention
One, REVHA1101 strain p30 gene PCR amplification
With pcr amplification REVHA1101 strain p30 gene, PCR primer is through 80V, after 1% agarose gel electrophoresis 1h, observes, as shown in Figure 1 under gel imaging system.In Fig. 1, M represents 1KbDNAMarker；The 1 pcr amplification result representing normal CEF cellular genome.2, represent that the pcr amplification result of CEF cellular genome is infected in REVHA1101 strain, size is about the band of about 627bp.
Two, REVp30 sequencing result and analysis thereof
Through Lasergene software, the sequencing result of pGEM-T-p30 positive colony is analyzed, whole purpose fragment includes whole p30 coding region, containing 627 nucleotide, encode 209 aminoacid, equally except U.S.'s SNV strain and China's HA9901 strain, this fragment homology in nucleic acid level and between other REV strains is all more than 99%；And on amino acid levels, compare with the p30 of other toxic strain, only become alanine at 89 amino acids places from threonine, and the homology between each strain has all reached 99.5%.
Three, the structure of pGEX-6p-1-p30 prokaryotic expression carrier
Take the correct pGEM-T-p30 of sequencing result and expression vector pGEX-6p-1 to use respectivelyEcoRI andXhoAfter I double digestion, after p30 and prokaryotic expression carrier pGEX-6p-1 is connected, transformed competence colibacillus e. coli bl21 (DE3), the single colony inoculation fluid medium of picking, SDS alkaline lysis method of extracting plasmid, then use respectivelyEcoRI andXhoI double digestion is identified, plasmid pGEX-6p-1-p30Digestion products occurs that size is about the purpose band of 627bp and size is about the band of 4.9Kb, is consistent with intended result.
Four, the abduction delivering of recombiant protein
Take the correct plasmid pGEX-6p-1-p30 of sequencing result and be transformed into e. coli bl21 (DE3), the plasmid pGEX-6p-1 being not inserted into exogenous sequences is transformed into the empty carrier bacterium of e. coli bl21 (DE3) gained simultaneously respectively in Amp+2 × YT fluid medium, add its final concentration of IPTG(and reach 1mM), 37 DEG C of abduction delivering 5h；Then collect bacterial precipitation respectively, collect the upper cleer and peaceful precipitation of cracking after ultrasonication respectively, carry out SDS-PAGE analysis.
In Fig. 2, M is non-pre-dyed protein standard；1 cracks supernatant for recombinant bacterium；2 is recombinant bacterium cracking precipitation；3 is non-recombinant bacterium induced product.
As seen from Figure 2: plasmid pGEX-6p-1-p30 is transformed into recombination fusion protein GST-p30 that e. coli bl21 (DE3) obtains afterwards after abduction delivering, the upper cleer and peaceful precipitation of its cracking all occurs in that a molecular weight is about the protein band of 49KD, and is not inserted in exogenous sequences recombinant bacterium and only occurs in that a molecular weight is about the GST label protein band of 27KD.
Five, the development of monoclonal antibody
Using above-mentioned recombination fusion protein GST-p30 as immunogen, immunity 6 week old Balb/c white mice, take its splenocyte with SP2/0 cell through twice fusion, obtain five strains altogether and can secrete the hybridoma cell strain of the monoclonal antibody reacted with GST-p30 but do not react with GST-Tag.Finding after testing, wherein the monoclonal antibody of two strain secretions all can be reacted with the p30 infected in the CEF pyrolysis product that REVHA1101 or SNV strain is infected, and fluorescence signal concentrates in cytoplasm (as shown in Figure 3) equally characteristically, is respectively designated as REVp30-2C8 and REVp30-5G6。
The preservation information of two strain monoclonal antibodies is respectively as follows:
REVp30The preserving number of-2C8 is: CGMCCNo.9218, and preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and China General Microbiological culture presevation administrative center, preservation date is on May 7th, 2014.
REVp30The preserving number of-5G6 is: CGMCCNo.9217, and preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and China General Microbiological culture presevation administrative center, preservation date is on May 7th, 2014.
Six, the determination of capture antibodies and enzyme labelled antibody combination
As below for REV-p30Double fastener heart combinations of pairs table shown in, in all different capture antibodies combinations of pairs with enzyme labelled antibody, with REVp30-2C8 is capture antibodies, REVp30-5G6EFor the combination of enzyme labelled antibody, under all different capture antibodies Concentraton gradient, OD450It is the highest, therefore determines with REVp30-2C8 is capture antibodies, REVp30-5G6EThe double sandwich-ELISA for REV group specific antigen p30 is set up for enzyme labelled antibody.
The exploration of seven, detection limit
According to the method described above, virus titer and the TCID of REVHA1101 or SNV are made50The variation relation figure of absorption value, as shown in Figure 4: double sandwich-ELISA method that the present invention sets up is minimum can detect 101.82Individual TCID50The p30 antigen entrained by REVHA1101 or SNV.
Eight, test kit detection application
React with the CEF product of cell lysis that REVHA1101, SNV, GST-p30, normal CEF cell, ALV-J, CAV, MDV or HVT are infected by double sandwich-ELISA method respectively that set up, obtain each TCID50The variation relation figure of absorption value, as shown in Figure 5.
Result shows: according to the above-mentioned positive criteria determined, the double sandwich-ELISA method that the present invention sets up only has positive reaction with REVHA1101, SNV and GST-p30, with all the other cause of diseases or normal CEF cell no cross reaction.

Claims (1)

1. the test kit of double sandwich-ELISA detection avian reticuloendotheliosis virus antigen, it is characterized in that: include capture antibodies and enzyme labelled antibody, wherein said capture antibodies is produced by hybridoma cell strain CGMCCNo.9218 secretion, described enzyme labelled antibody is produced by hybridoma cell strain CGMCCNo.9217 secretion, and the antigen of test kit detection is REVP30 proteantigen.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5130232A (en) * 1990-06-12 1992-07-14 The United States Of America As Represented By The Secretary Of Agriculture Monoclonal antibody immunoassay kit for avian reticuloendotheliosis virus
CN102031245A (en) * 2010-10-19 2011-04-27 中国农业科学院哈尔滨兽医研究所 Monoclonal antibody of avian reticuloendothelial hyperplasia protein gp90 and application thereof
CN102680682A (en) * 2012-05-30 2012-09-19 山东农业大学 Kit for detecting antibody of reticuloendotheliosis virus (REV) through enzyme-linked immunosorbent assay (ELISA)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5130232A (en) * 1990-06-12 1992-07-14 The United States Of America As Represented By The Secretary Of Agriculture Monoclonal antibody immunoassay kit for avian reticuloendotheliosis virus
CN102031245A (en) * 2010-10-19 2011-04-27 中国农业科学院哈尔滨兽医研究所 Monoclonal antibody of avian reticuloendothelial hyperplasia protein gp90 and application thereof
CN102680682A (en) * 2012-05-30 2012-09-19 山东农业大学 Kit for detecting antibody of reticuloendotheliosis virus (REV) through enzyme-linked immunosorbent assay (ELISA)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
An enzyme-linked immunosorbent assay for detection of reticuloendotheliosis virus infection in chickens.;J.Ignjatovic, et al.;《Avian Pathology》;19871231;第16卷(第4期);摘要，第611页第22-44行，第612页第24-41行，表2 *
禽网状内皮组织增生症病毒p30蛋白的表达及检测;张石磊，等;《中国动物传染病学报》;20111130;第19卷(第6期);第15-19页 *

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