CN104330560A - Kit for detecting antigen of avian reticuloendotheliosis virus (REV) by dual-sandwich ELISA and application of kit - Google Patents

Abstract

The invention discloses a kit for detecting an antigen of an avian reticuloendotheliosis virus (REV) by dual-sandwich ELISA, belonging to the field of detection of biotechnology. The kit is used for detecting a REVP 30 protein antigen and can minimally detect P30 antigens carried by 10<1.82> REVHA1101 or SNV viral strains of TCID50. The kit provides a reliable means for rapidly detecting the REV antigen clinically. The kit is simple, convenient and rapid to operate, can be used for detecting the REV, and is suitable for veterinarian departments of all stages in the primary level, entry and exit inspection and quarantine, vaccine manufacture enterprises and the like to rapidly detect REVs in large scale. The method is low in required cost, remarkable in economic benefit and wide in application prospect.

Description

Double sandwich-ELISA detects avian reticuloendotheliosis viral antigen kit and application thereof

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 inhibitive ability of immunity disease, and its harm caused poultry farming 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) is in the infection of poultry and the popular trend having increase, and the mixed infection between other tumorigenic disease often major injury fowl group productive capacity and cause certain economic loss, adverse effect is caused to poultry farming.

Since 1958, REV is propagated in the fowl group of multiple countries and regions by number of ways, presents worldwide distribution.According to the Serologic detection display carried out in Korea S, Egypt, Taiwan and the U.S. in recent years, all there is REV antibody positive in multiple chicken group and turkey group, positive rate is not from 34% to 92% 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 sick epidemiology survey, in a lot of chicken group, finds that there is higher REV antibody positive rate.PCR and antigen detection display, the REV all existed in broiler chicken, laying hen, aquatic bird in various degree infects, and in the chicken group of China mainland area, the recall rate of REV, is the highest in all detection samples.

The situation that REV and other several immunosuppressive diseases comprise MDV, ALV and CAV mixed infection is very general, is to cause chicken group to occur sever immune suppression, strengthen one of pathogenic major reason of other cause of diseases.The chicken group of ALV-J and REV coinfection, although do not have obvious suppression for the immune response of REV, but then can seriously suppress for the immune response of ALV-J, and the duration is 7 weeks, have a strong impact on the productive capacity of chicken group, also hamper to the work of ALV-J eradication, under experimental conditions, the mixed infection of REV and MDV can reduce the ability that body produces IFN-γ, inhibits body for the innate immunity ability of virus.

In addition, the insertion of REV viral nucleic acid in other viral genome is also one of emphasis of academia's concern 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 large section of the REV genome of LTR to insert its viral DNA genomic phenomenon.In bird pox virus, compare with the bird pox virus not inserting REV fragment or Insert Fragment negligible amounts, insert replication capacity in host of REV genome a fairly large number of bird pox virus and virulence has obvious enhancing.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 obviously weakens, being inserted in the communication process of MDV of prompting REV fragment is significant.

The separation andpreconcentration of cause of disease is the most classical, the most reliable REV detection method, the internal organs lapping liquid of general use bird 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, and then use the monoclonal antibody for REV carry out immunofluorescence, SABC, complement fixation test etc. to may exist in cell culture virus detect.This process length consuming time, and sensitivity, simplicity are also strong, but reliable results, usually as the goldstandard of other detection methods with for referencial use.Sandwich ELISA based on monoclonal antibody or polyvalent antibody or antibody capture ELISA method can direct REV env or p to comprising in enormous quantities in the suspicious sample of anus swab and egg white at short notice ³⁰antigenic component detects, and without the need to carrying out virus amplification in advance on permissive cell, simple and effective, economy are convenient, have very high use value.But more under experimental conditions, still there is higher false positive rate in the ELISA detection method based on resisting, and adopts monoclonal antibody to address this problem.

Serologic detection, can adopt multiple method to detect REV antibody or antigen in Suspect serum, egg white or yolk, but its frequency occurred and duration inconsistent.Can be antigen with the permissive cell of fixing infection REV, IFA detection is carried out to Suspect serum sample, also can with viral Neutralizing test, agar gel precipitation experiment (AGP) or with gp ⁹⁰for the indirect ELISA of antigen or stop band restrain experiment etc. detect, wherein agar gel precipitation experiment can be used for the detection of antigen and antibody but sensitivity is not high.

Therefore need at present that exploitation is efficient, quick, convenient, financial cost is lower 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 kit that a kind of double sandwich-ELISA detects avian reticuloendotheliosis viral antigen, to adapt to the diagnosis needs efficient, quick, convenient, financial cost is lower.

The present invention includes and catch antibody REVp ³⁰-2C8 and enzyme labelled antibody REVp ³⁰-5G6.

Through qualification, the P30 in the CEF pyrolysis product that these monoclonal antibodies all can infect with REV HA1101 strain and SNV strain reacts, and does not react with the CEF product of cell lysis of the virus infections such as normal CEF cell, ALV-J, CAV, MDV or HVT.The kit that the present invention sets up, adopts that ELISA method is minimum can detect 10 ^1.82individual TCID ₅₀the P30 antigen entrained by REV HA1101 or SNV.Adopt this 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.

Another object of the present invention proposes the application that double sandwich-ELISA detects avian reticuloendotheliosis virus (REV) antigenic reagent box.

The present invention adopts ELISA detection method, feature is: cell culture to be checked, cloaca swab or egg white are done, and the REV P30 recombinant products equal samples of vivoexpression, after suitable dilution, add in this kit ELISA Plate, the P30 antigen whether containing REV or REV in sample can be detected.

Adopt above method, can P30 antigen fast and efficiently whether containing avian reticuloendotheliosis virus (REV) or REV in judgement sample.

Accompanying drawing explanation

Fig. 1 is the gel electrophoresis analysis figure of PCR amplification REV HA1101 strain p30 gene.

Fig. 2 is the abduction delivering analysis chart of recombinant protein.

Fig. 3 is REVp ³⁰-2C8 and REVp ³⁰the Monoclonal Antibody Cell supernatant IFA result picture of-5G6.

Fig. 4 is virus titer and the Detection results graph of a relation that kit detects REV HA1101 or SNV.

Fig. 5 is the reaction result figure that kit detects different virus or vivoexpression product.

Embodiment

one, REV HA1101 strain p30 gene PCR amplification

With pcr amplification REV HA1101 strain p30 gene, PCR primer, through 80V, after 1% agarose gel electrophoresis 1h, is observed, as shown in Figure 1 under gel imaging system.In Fig. 1, M represents 1Kb DNA Marker; The pcr amplification result of the normal CEF cellular genome of 1 expression.2, represent that the pcr amplification result of CEF cellular genome is infected in REV HA1101 strain, size is about the band of about 627bp.

two, REV p30 sequencing result and analysis thereof

Analyze through the sequencing result of Lasergene software to pGEM-T-p30 positive colony, whole object fragment comprises whole p30 code area, containing 627 nucleotide, to encode 209 amino acid, same except U.S. SNV strain and Chinese HA9901 strain, the homology of this fragment 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 all reaches 99.5%.

three, the structure of pGEX-6p-1-p30 prokaryotic expression carrier

Get the correct pGEM-T-p30 of sequencing result and expression vector pGEX-6p-1 to use respectively ecoRi and xhoafter I double digestion, after p30 is connected with prokaryotic expression carrier pGEX-6p-1, transform competent E. coli BL21 (DE3), the single colony inoculation fluid nutrient medium of picking, SDS alkaline lysis method of extracting plasmid, then use respectively ecoRi and xhoi double digestion is identified, plasmid pGEX-6p-1-p ³⁰digestion products occurs conforming to the band that a size is about the object band of 627bp and size and is about 4.9Kb with intended result.

four, the abduction delivering of recombinant protein

Get the correct plasmid pGEX-6p-1-p30 of sequencing result and be transformed into e. coli bl21 (DE3), the plasmid pGEX-6p-1 not inserting exogenous sequences is transformed into the empty carrier bacterium of e. coli bl21 (DE3) gained respectively in Amp+ 2 × YT fluid nutrient medium simultaneously, add its final concentration of IPTG(and reach 1mM), 37 DEG C of abduction delivering 5h; Then collect bacterial precipitation respectively, collect cleer and peaceful precipitation in cracking after ultrasonication respectively, carry out SDS-PAGE analysis.

In Fig. 2, M is non-pre-dyed protein standard; 1 is recombinant bacterium cracking supernatant; 2 is recombinant bacterium cracking precipitation; 3 is non-recombinant bacterium induced product.

As seen from Figure 2: the recombination fusion protein GST-p30 that plasmid pGEX-6p-1-p30 obtains after being transformed into e. coli bl21 (DE3) is after abduction delivering, its cracking all occurs in cleer and peaceful precipitation a molecular weight is about the protein band of 49KD, and do not inserted in exogenous sequences recombinant bacterium and only occurred 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 immunogene, immunity Balb/c small white mouse in 6 week age, get its splenic lymphocyte and SP2/0 cell through twice fusion, obtain five strains altogether and can secrete and react but the hybridoma cell strain of the monoclonal antibody of not reacting with GST-Tag with GST-p30.Find after testing, the monoclonal antibody of wherein two strains secretions all can react with the p30 infected in CEF pyrolysis product that REV HA1101 or SNV strain infect, and fluorescence signal concentrates on cytoplasm interior (as shown in Figure 3) equally characteristically, distinguishes called after REVp ³⁰-2C8 and REVp ³⁰-5G6.

The preservation information of two strain monoclonal antibodies is respectively:

REVp ³⁰the preserving number of-2C8 is: CGMCC No.9218, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China General Microbiological culture presevation administrative center, and preservation date is on May 7th, 2014.

REVp ³⁰the preserving number of-5G6 is: CGMCC No.9217, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China General Microbiological culture presevation administrative center, and preservation date is on May 7th, 2014.

six, the determination of antibody and enzyme labelled antibody combination is caught

As following for REV-p ³⁰double fastener heart combinations of pairs table shown in, catch in the combinations of pairs of antibody and enzyme labelled antibody, with REVp in all differences ³⁰-2C8 is for catching antibody, REVp ³⁰-5G6 ^efor the combination of enzyme labelled antibody, under all differences catch antibody concentration gradient, OD ₄₅₀be the highest, therefore determine with REVp ³⁰-2C8 is for catching antibody, REVp ³⁰-5G6 ^efor enzyme labelled antibody sets up the double sandwich-ELISA for REV group specific antigen p30.

seven, the exploration of detectability

According to the method described above, virus titer and the TCID of REV HA1101 or SNV is made ₅₀the variation relation figure of absorption value, as shown in Figure 4: the double sandwich-ELISA method that the present invention sets up is minimum can detect 10 ^1.82individual TCID ₅₀the p30 antigen entrained by REV HA1101 or SNV.

eight, kit detects application

Respectively the CEF product of cell lysis that REV HA1101, SNV, GST-p30, normal CEF cell, ALV-J, CAV, MDV or HVT infect is reacted with the double sandwich-ELISA method set up, obtain each TCID ₅₀the 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 REV HA1101, SNV and GST-p30, with all the other cause of diseases or normal CEF cell no cross reaction.

Claims (3)

1. double sandwich-ELISA detects avian reticuloendotheliosis viral antigen kit, it is characterized in that: comprising seizure antibody is REVp ³⁰-2C8 and enzyme labelled antibody are REVp ³⁰-5G6.

2. kit according to claim 1, is characterized in that: the antigen that kit detects is REV P30 proteantigen.

3. double sandwich-ELISA detects the application of avian reticuloendotheliosis viral antigen kit as claimed in claim 1, it is characterized in that: after the P30 recombinant products of cell culture to be checked, cloaca swab, egg white or vivoexpression is diluted, add in this kit ELISA Plate, for detecting the P30 antigen whether containing REV or REV in sample to be checked.