CN116218912A - ELISA kit for diagnosing APPV antibody by using mammalian cell line for stably expressing APPV E2 protein - Google Patents

ELISA kit for diagnosing APPV antibody by using mammalian cell line for stably expressing APPV E2 protein Download PDF

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CN116218912A
CN116218912A CN202211592113.6A CN202211592113A CN116218912A CN 116218912 A CN116218912 A CN 116218912A CN 202211592113 A CN202211592113 A CN 202211592113A CN 116218912 A CN116218912 A CN 116218912A
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appv
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仇华吉
罗玉子
宋浩
高晓薇
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Harbin Veterinary Research Institute of CAAS
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Abstract

The invention discloses a mammalian cell line for stably expressing APPV E2 protein and an ELISA kit for diagnosing APPV antibody. The invention firstly constructs a mammal suspension cell line for stably expressing APPV E2 protein, successfully obtains recombinant APPV E2 protein with high expression level, high purity and good antigenicity, and greatly reduces the production cost. On the basis, an indirect ELISA kit for diagnosing the atypical pestivirus antibody of the pig is established by adopting the expressed recombinant APPV E2 protein, has high sensitivity and specificity, has no cross reaction with positive serum of other virus antibodies, has the coincidence rate of 93.7% with VNT, and can be used for the specificity detection of the APPV antibody; on the basis, serum epidemic rates of APPV in pig groups are detected by using the established kit by collecting pig serum samples from 9 provinces in China, and the result shows that the total serum epidemic rate of APPV is 73.9%.

Description

ELISA kit for diagnosing APPV antibody by using mammalian cell line for stably expressing APPV E2 protein
Technical Field
The invention relates to a cell line for expressing an E2 protein of a swine atypical pestivirus (atypical porcine pestivirus, APPV) and an ELISA kit for diagnosing an APPV antibody, in particular to a mammalian suspension cell line for stably expressing the APPV E2 protein and an ELISA kit for diagnosing the APPV antibody, belonging to the field of diagnosis of the swine atypical pestivirus.
Background
Pestiviruses belong to the flaviviridae family and include a number of important pathogens that seriously jeopardize the healthy development of the animal industry, causing significant economic losses to the breeding industry, such as classical swine fever virus (classical swine fever virus, CSFV) and bovine viral diarrhea virus (bovine viral diarrhea virus, BVDV). Pestiviruses are growing in variety year by year, and currently pestiviruses comprise at least 11 species (pestiviruses a to K). Porcine atypical pestiviruses (atypical porcine Pestivirus, APPV) were found in 2015 by american scientists by metagenomic sequencing from Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) genome-positive porcine serum samples and were classified as Pestivirus K (Hause, b.m., collin, e.a., pedirieddi, l., yuan, f., chen, z, hesse, r.a., gauge, p.c., clement, t., fang, y,&anderson, g. (2015) Discovery of a novel putative atypical porcine pestivirus in pigs in the usa. Journal of General Virology,96,2994-2998.Https:// doi. Org/10.1099/jgv.0.000251). Subsequently, by two independent studies of vaccinating pregnant sows with infectious animal material, it was demonstrated that congenital tremor of newborn piglets is associated with the presence of APPV. Like other pestiviruses such as CSFV, BVDV and borderline disease virus (border disease virus, BDV), the APPV genome is a linear, non-segmented, sense RNA, approximately 11 to 12kb in size, comprising a large open reading frame (open reading frame, ORF). The ORF encodes a multimeric protein consisting of about 3,635 amino acids, in itself and in the host proteinProcessing and hydrolysis by enzymes to produce four structural proteins (C, E) rns E1, E2) and eight nonstructural proteins (N pro P7, NS2, NS3, NS4A, NS4B, NS a, and NS 5B) (Hause, b.m., collin, e.a., pedirieddi, l., yuan, f., chen, z., hesse, r.a., gauge, p.c., clement, t., fang, y.,&anderson, g. (2015) Discovery of a novel putative atypical porcine pestivirus in pigs in the usa. Journal of General Virology,96,2994-2998.Https:// doi. Org/10.1099/jgv.0.000251). APPV has 68% amino acid homology with batwing pestiviruses, but less than 40% amino acid homology with CSFV, BVDV, and BDV, based on the polyprotein amino acid sequence (Hause et al, 2015). Furthermore, the data obtained at present provide no evidence that the presence of APPV genome or APPV-specific antibodies would interfere with the detection methods conventionally used for CSF diagnosis (poltel, a., meyer, d., petrov, a.,&Becher,P.(2017a).Recent emergence of a novel porcine pestivirus:interference with classical swine fever diagnosis Emerging Microbes&Infections,6(4),e19.https://doi.org/10.1038/emi.2017.5)。
APPV is widely distributed throughout the world, and is detected in pigs in north america and south america, europe and asia. The mortality rate of APPV infected piglets is high, about 30% in some affected pig farms, severely affecting the economic development of the pig industry. APPV was first reported in the province of guangdong in 2016 and subsequently detected in the south and north of china, including Sichuan, guangxi, anhui, hubei, henan, etc.
The widespread distribution of APPV-infected pigs across different geographical areas of the world, coupled with the increase in international commerce and travel, greatly increases the risk of APPV transmission to non-endemic areas. Thus, there is an urgent need to develop economically viable, stable, sensitive and specific diagnostic methods to detect potential APPV infections in susceptible hosts, especially in non-endemic areas. To date, several Polymerase Chain Reactions (PCRs) have been reported for detecting APPV nucleic acids, including reverse transcription real-time quantitative PCR (RT-qPCR), drop digital PCR (ddPCR) and multiplex RT-qPCR. Given that antibodies appear early in viral infection, which can last months or even years, and that there is no commercial APPV vaccine at present, serological tests are more able to assess the infection status (infected or uninfected) of animals than nucleic acid tests. For pestiviruses, the virus neutralization assay (virus neutralization test, VNT) is a gold standard for detecting antiviral antibodies. However, VNT requires a biosafety laboratory capable of handling live viruses and is too time consuming and complex to be suitable for clinical field detection. Furthermore, APPV strains are more difficult to isolate, preventing the use of VNT in diagnostics.
Indirect enzyme-linked immunosorbent assay (iELISA) is widely used throughout the world due to its simplicity, high throughput, safety and rapidity, however, the diagnostic performance of iELISA is affected by the quality of its expressed antigen. There is no commercial APPV serodiagnostic method at present, and the diagnostic performance (sensitivity, specificity and reproducibility) of the ELISA for detection of APPV structural protein antibodies described previously has not been strictly evaluated (Schwarz, l., riedel, c., hogler, s, sinn, l.j., vogmayr, t., wochtl, b., dinhopl, n., rebel-Bauder, b., weissenbock, h., landig, a., chumendorf, t., and & Lamp, b. (2017), congenital infection with Atypical Porcine Pestivirus (APPV) is associated with disease and viral persistence.velrinary Research,48,14.Https:// doi.org/10.1186/s 13567-016-0406-1).
Given the widespread worldwide popularity of APPV and the serious threat to newborn piglets in pig farms, together with the fact that the pathogen becomes a recessive infection in adult pigs, it is highly cryptic to understand the epidemic situation of this virus and to effectively diagnose APPV infection is crucial for disease control. The key to developing a specific ELISA method for detecting viral antibodies is the selection of the appropriate viral antigen. After pestivirus infection, the virus can generate the virus against E2 and E rns And antibodies to NS3 protein. Neutralizing antibodies conferring protective immunity are mainly induced by E2, so serological diagnosis of pestiviruses is mainly based on detection of E2 specific antibodies. However, the E2 protein is an envelope glycoprotein with a highly glycosylated modification, and studies have shown that loss of the protein glycosylated modification may affect immunogenicity (Arruda, b.l., falkenberg, s., mora-daz, j.c., mattas ferrey ra, f.s., magtoto, r.,&Giménez-Lirola,L.(2022).Development and evaluation ofanti-specific dual matrix Pestivirus K ELISAs using longitudinal known infectious status samples. Journal of Clinical Microbiology, e0069722.Https:// doi. Org/10.1128/jcm. 00697-22). In view of the advantages of eukaryotic expression systems such as protein processing, folding and post-translational modification, which are not possessed by prokaryotic systems, the eukaryotic expression systems are more favorable for the expression of envelope glycoprotein E2 with high glycosylation modification, and the biological activity of the protein is ensured. Therefore, the adoption of a eukaryotic expression system for stably and efficiently expressing the APPV E2 protein is of great importance for developing an APPV serodiagnosis kit which can be applied commercially.
Disclosure of Invention
It is an object of the present invention to provide the construction of a mammalian suspension cell line stably expressing the APPV E2 protein and recombinant APPV E2 protein expressed by the mammalian suspension cell line;
the second object of the invention is to provide an ELISA kit for diagnosing or detecting the atypical swine fever virus;
the above object of the present invention is achieved by the following technical solutions:
in one aspect, the invention provides a method of constructing a mammalian suspension cell line stably expressing an APPV E2 protein, comprising:
(1) Subcloning the plasmid containing APPV E2 gene into slow virus expression vector to obtain recombinant expression plasmid;
(2) Co-transfecting the recombinant expression plasmid and the auxiliary plasmid into HEK293T cells, and then harvesting recombinant lentivirus;
(3) And (3) transducing the harvested recombinant lentivirus into HEK293 suspension cells to obtain a HEK293-APPV-E2 suspension cell line for stably expressing the APPV E2 protein.
As a preferred embodiment of the method of constructing a mammalian suspension cell line stably expressing the APPV E2 protein, the APPV E2 gene is optimized according to the synonymous codon bias of the mammal in step (1); furthermore, ecoRI restriction enzyme cutting sites, kozak sequences and signal peptides are introduced into the N end of the optimized APPV E2 gene, and thrombin cutting sites, strep tags, 6 XHis tags and XhoI restriction enzyme cutting sites are introduced into the C end.
As a preferred embodiment of the method for constructing a mammalian suspension cell line stably expressing APPV E2 protein, a plasmid containing the APPV E2 gene is subcloned into a lentiviral expression vector pLVX-IRES-ZsGreen1 in step (1) to obtain a recombinant expression plasmid pLVX-APPV-E2.
As a preferred embodiment of the method for constructing a mammalian suspension cell line stably expressing APPV E2 protein, the recombinant plasmid pLVX-APPV-E2 and helper plasmids pMD2.G and psPAX2 are co-transfected into HEK293T cells in step (2) and then the recombinant lentivirus is harvested.
The invention adopts the suspension HEK293 cells to express the recombinant APPV E2 protein, and the HEK293 cells are easy to transfect, can adapt to suspension culture and growth, and can achieve high-efficiency expression of the APPV E2 protein. The mammalian cell expression system is used for APPV E2 protein expression, and compared with prokaryotic expression, the mammalian cell expression system for expressing exogenous protein can retain the functional activity of viral protein, so that the expressed protein structure is similar to that of natural protein. In addition, the HEK293-APPV-E2 suspension cell line for stably expressing the APPV E2 protein constructed by the invention meets the development requirement of a commercial ELISA kit and has the characteristics of convenience and high yield.
Another aspect of the invention is the use of recombinant APPV E2 protein expressed by a mammalian suspension cell line stably expressing APPV E2 protein in the preparation of reagents for diagnosing or detecting APPV antibodies.
As a preferred embodiment, for example, the recombinant APPV E2 protein thus prepared can be used as a coating antigen for preparing diagnostic reagents for detection of APPV antibodies.
Accordingly, the present invention provides an Indirect ELISA (iELISA) kit for diagnosing or detecting an antibody against a swine atypical pestivirus, comprising: coating antigen, primary antibody, enzyme-labeled secondary antibody, antibody diluent, washing solution, sealing solution, color development solution and stop solution; wherein the coating antigen is recombinant APPV E2 protein expressed by a mammalian suspension cell line which stably expresses the APPV E2 protein.
The invention optimizes the reaction parameters of the established indirect ELISA kit for diagnosing or detecting the atypical pestivirus antibody of the pig, and uses the optimized method to detect and analyze 165 parts of APPV antibody negative serum determined by virus neutralization test (virus neutralization test, VNT), thereby determining the critical value to be 0.2. The invention further provides the repeatability, sensitivity and specificity of the ELISA kit for diagnosing or detecting the atypical swine fever virus. Evaluation of pig serum samples confirmed by VNT using established indirect ELISA kit showed high sensitivity (113/120, 94.2%) and specificity (65/70, 92.9%), no cross reaction with antibody positive serum of PRRSV, CSFV, BVDV, PRV and PCV2, 93.7% compliance with VNT (178/190), and can be used for specific detection of APPV antibodies; the repeatability experiment shows that the CV of the ELISA kit provided by the invention is less than 10% in batches and between batches; based on the detection, 1368 pig serum samples are collected from intensive pig farms of 9 provinces in China, and the established indirect ELISA kit is used for detecting the serum prevalence rate of APPV in pig groups, and the result shows that the total serum prevalence rate of APPV is 73.9% (1011/1368), which indicates that APPV is popular in multiple areas in China.
Drawings
FIG. 1 expression of APPV E2 protein in mammalian suspension HEK293 cells; (a) suspending HEK293 cells; following transduction, HEK293-APPV-E2 expression.
FIG. 2Western blotting and SDS-PAGE to verify the expression of the recombinant protein; (A) Western blotting analysis results are carried out on the recombinant E2 protein expression condition by using an anti-His monoclonal antibody; m: protein marker;1: suspension HEK293 cell culture supernatant of transfected empty vector; 2: HEK293-APPV-E2 suspension cell line culture supernatant; (B) SDS-PAGE analysis of the purified recombinant E2 protein; m: protein marker;1: suspension HEK293 cell culture supernatant of transfected empty vector; 2: purified recombinant E2 protein.
FIG. 3E2-iELISA results of optimization of working conditions; (A) Determining the concentration of the coating antigen and the serum dilution by adopting a chessboard titration method; (B) And (C) is the optimized result of the dilution of the second enzyme-labeled antibody and the reaction time of TMB; (D) And (E) optimizing the results for the incubation time of the serum sample and the enzyme-labeled secondary antibody.
FIG. 4 shows the evaluation results of E2-iELISA; (A) determination of E2-iELISA threshold; the threshold was determined from an APPV antibody negative serum sample (n=165); APPV antibody positive serum (n=62) as control; (B) Pig serum of known anti-APPV NAb titer was serially diluted in gradient for evaluation of sensitivity of E2-iielisa; n1, APPV antibody negative serum; p1, APPV antibody positive serum; numbers in brackets indicate NAb titer; (C) The specificity of E2-iielisa was verified by a panel of porcine antisera against non-APPV virus, including BVDV (n=4), PRV (n=5), PCV2 (n=3), PRRSV (n=2) and CSFV (n=4).
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Plasmid, cell, virus and serum samples used in the following examples and test examples
Lentiviral vector pLVX-IRES-ZsGreen1 and helper plasmid psPAX2, pMD2.G were purchased from Addgene corporation (USA). Human Embryonic Kidney (HEK) 293T cells and porcine kidney (PK-15) cells were cultured in DMEM medium containing 10% fetal bovine serum, 100. Mu.g/mL streptomycin and 100IU/mL penicillin at 37℃and 5% CO 2 Culturing under the condition. Suspension HEK293 cells in serum free
Figure BDA0003995079660000071
The cells were cultured in CD 293M medium (Basaimia, china).
APPV China/HLJ491/2017 strain was isolated by homogenate inoculation of APPV RT-PCR positive porcine tissue into PK-15 cells (unpublished data). BVDV antibody positive serum used in this study was supplied by OIE swine fever reference laboratory, university of veterinary medicine, hanou, germany; all APPV antibody positive and negative sera were kept by the present laboratory; other relevant porcine antisera useful for evaluating the specificity of the present study method were maintained by the present inventors laboratories, including PRRSV, CSFV, porcine circovirus type 2 (PCV 2) and porcine pseudorabies virus (PRV).
Statistical analysis method used in the following test examples
All data were analyzed using GraphPad Prism software.
EXAMPLE 1 eukaryotic expression, purification and characterization of the APPV E2 protein
1 test method
1.1 construction of recombinant plasmids
The E2 coding region sequence of the APPV China/HLJ491/2017 strain (GenBank accession number OP 617199) was optimized according to synonymous codon bias of the mammal. Then, an EcoRI restriction enzyme cutting site, a Kozak sequence and a signal peptide (Kanekiyo et al, 2013) are introduced into the N end of the optimized E2 gene, and a thrombin cutting site, a Strep tag, a 6 XHis tag and an XhoI restriction enzyme cutting site are introduced into the C end at the same time, so that the designed sequence is sent to the Huada gene for gene synthesis. Subsequently, the synthesized plasmid pMV-APPV-E2 was subcloned into a lentiviral expression vector pLVX-IRES-ZsGreen1 to obtain a recombinant expression plasmid pLVX-APPV-E2. All plasmids were verified by sequencing.
1.2 construction of HEK293 suspension cell lines stably expressing APPV E2 protein
For high level expression of recombinant E2 protein, HEK293 suspension cell line stably expressing APPV E2 protein was constructed using lentiviral expression system. Normally grown HEK293T cells were plated in 10cm dishes and co-transfected with recombinant plasmid pLVX-APPV-E2 and helper plasmids pMD2.G and psPAX2 until confluency was about 90%. At 48h post-transfection, recombinant lentiviruses were harvested and transduced into HEK293 suspension cells to obtain HEK293-APPV-E2 suspension cell lines. Expression of recombinant E2 protein in transduced cells was detected by Western blotting analysis. And further carrying out continuous passage on the HEK293-APPV-E2 suspension cell line for at least 20 generations, and verifying the stable expression condition of the recombinant protein.
1.3Western blotting
Western blotting assays (Ji, S, luo, Y, zhang, T, shao, L, meng, X.Y., wang, Y, gao, Y, li, S, sun, Y, jin, X, & Qiau, H.J. (2018) An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells of biology, 163 (7), 1831-1839.Https:// doi.org/10.1007/s 00705-018-3809-7) were performed as described previously. Briefly, cell culture supernatants collected 96h post transduction were incubated overnight with Ni-NTA High Performance resin, followed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, protein strips were transferred onto nitrocellulose membranes with a semi-dry transfer membrane (Bio-Rad), then the membranes were blocked for 2h in blocking buffer (PBS containing 5% skim milk), after washing off the skim milk with PBS-Tween 20 (PBST), specific anti-His tag antibodies (1:1000) were added and acted in a horizontal shaker for 2h; the membranes were then washed four times with PBST and incubated with IRDye 800CW secondary antibody (1:10 000) at room temperature in the absence of light for 1h. Finally, the films were scanned using an Odyssey infrared fluorescence scanning imaging system (Li-Cor Biosciences) and the results analyzed.
1.4 purification of recombinant E2 proteins
HEK293-APPV-E2 cell line for 8 days, the supernatant was collected and filtered through a 0.45 μm pore size filter. Ni was performed according to the Ni-NTA High Performance (GE Healthcare, USA) operating manual 2+ The recombinant protein was purified by affinity chromatography and subjected to SDS-PAGE analysis, and protein bands were observed after coomassie blue staining, and protein concentration was determined using BCA assay kit.
2 test results
Lentiviral expression vector pLVX-IRES-ZsGreen1 allows for simultaneous expression of foreign protein and Green Fluorescent Protein (GFP) in mammalian cells, thus green fluorescence was observed under an inverted microscope after successful construction of pLVX-APPV-E2 plasmid transduction of suspension HEK293 cells (fig. 1). Compared with the pLVX-IRES-ZsGreen1 empty vector, the HEK293-APPV-E2 cell line constructed by Western blotting detection shows that the specific protein is expressed after 4 days of culture. The results showed that the APPV E2 protein had a molecular weight of about 40 kDa (FIG. 2A), the recombinant protein was purified by affinity chromatography from the supernatant obtained after 8 days of culture of HEK293-APPV-E2 cell line, and that a diffuse band was observed around 40-55kDa by SDS-PAGE, presumably as a result of various degrees of glycosylation modification of the protein (FIG. 2B).
The HEK293-APPV-E2 suspension cell line obtained by the test is continuously transferred for 20 generations, and the recombinant E2 protein can still be stably and efficiently expressed, which shows that the HEK293 suspension cell line capable of stably expressing the recombinant APPV E2 protein is successfully obtained.
Experimental example 1 development and optimization of an indirect ELISA based on recombinant E2 protein
1 test method
To establish an efficient indirect ELISA diagnostic method, checkerboard titration was used to determine the optimal concentration of coating antigen and serum dilution. Briefly, purified recombinant E2 protein was coated on an ELISA plate at a concentration of 0.625 to 10. Mu.g/mL, and APPV antibody positive and negative sera verified by VNT were diluted at 1:50 to 1:400. Then, HRP-labeled rabbit anti-pig IgG was diluted 1:5000 to 1:40,000 to determine the optimal dilution of enzyme-labeled antibody, and two replicates were made for each sample. Purified recombinant E2 protein was diluted to the appropriate concentration with 0.05mol/L carbonate buffer (ph=9.6), 100 μl per well of coated elisa plate, incubated overnight at 4 ℃. Then 180. Mu.L of commercial blocking solution (Surmotics, USA) was added and blocked for 2h at 37 ℃. Then, the serum was diluted 1:100 with StabilZyme SELECT Stabilizer dilution (Surmotics, USA) and added to the blocked ELISA plate, incubated at 37℃for 1h, washed 4 times with PBST, and dried by pipetting; horseradish peroxidase (HRP) -labeled rabbit anti-pig IgG (Sigma-Aldrich, USA) was diluted 1:20000 with StabilZyme SELECT Stabilizer dilution, incubated for 1h at 37℃and washed 5 times with PBST, patted dry, added 100. Mu.L TMB chromogenic solution (Sigma-Aldrich, USA) and finally added 50. Mu.L 2M H 2 SO 4 The reaction was terminated and read using a microplate reader (ELx 808, bioTek) at a wavelength of 450 nm.
Virus neutralization assay and indirect immunofluorescence assay
APPV antibody positive and negative sera were determined by VNT (Luo et al, 2017). Briefly, serum was heat-inactivated at 56℃for 30min, and then clinical serum samples were serially diluted 10-fold (starting from 1/10) with DMEM. Diluted sample and equal volume of 200TCID 50 Virus (APPV) is mixed and inIncubate at 37℃for 1h. The antibody virus mixture was then added to a 96-well plate inoculated with PK-15 cells and incubated at 37 ℃ for 72h. Serum neutralizing antibody titers were then determined by IFA. 96-well plates were fixed with pre-chilled absolute ethanol at 4 ℃ for 30min, after which APPV anti-E2 monoclonal antibodies (diluted with 5% Bovine Serum Albumin (BSA) 1:200) were used as primary antibodies and incubated with cells for 2h at 37 ℃. Followed by 5 washes with PBST and incubation with FITC-labeled sheep anti-mouse IgG (Invitrogen) for 1h (diluted with 5% BSA 1:300) at 37 ℃. Finally, after 4 washes with PBST, 50% glycerol was added to each well and the results were recorded under a fluorescence microscope (Nikon TE200, japan). Serum neutralizing antibodies (neutralizing antibody, NAb) titres against APPV are expressed as the reciprocal of the highest serum dilution that inhibited viral infection (Reed and Muench, 1938).
Evaluation of Indirect ELISA
Cutoff value determination:
the Cutoff values of E2-iielisa were determined as described previously, with some modifications (Li et al, 2015). Briefly, 165 APPV antibody negative sera were tested using the established E2-iielisa as described above, and the S/P value of the negative sera was calculated = (sample OD to be tested) 450nm Average OD of negative samples 450nm ) (average OD of positive samples) 450nm Average OD of negative samples 450nm ) And Standard Deviation (SD), negative serum threshold = negative serum mean S/P value +3sd.
Sensitivity test:
1 part of APPV antibody positive serum and 1 part of APPV antibody negative serum are randomly picked, neutralization titer is measured by a neutralization test, and after serum samples are subjected to double-ratio dilution (1:100-1:12800), ELISA method established by the study is used for detection, and the sensitivity of the method is evaluated.
Specificity test:
to determine the specificity of the established indirect ELISA method, antibody positive sera to CSFV, BVDV, PRRSV, PCV2 and PRV were tested.
Repeatability test:
3 APPV antibody positive sera and 1 negative serum were randomly selected for detection, and the intra-and inter-batch Coefficient of Variation (CV) was calculated.
Compliance test:
190 pig blood serum with known background is detected by using established indirect ELISA method, and compared with the neutralization test result.
2 test results
Optimization of 2.1E2-iELISA
The E2-iELISA working conditions were optimized. The optimal working conditions are shown in FIG. 3, the concentration of the coating antigen is 2.5 mug/mL, the dilution of the serum sample is 1:100, and the dilution of the enzyme-labeled secondary antibody is 1:20,000. The optimal reaction times for serum, enzyme-labeled secondary antibody and TMB solution were 45, 45 and 10min, respectively.
Performance of 2.2E2-iELISA
To determine the threshold of E2-iELISA, 165 VNT-validated APPV antibody negative serum samples were tested with E2-iELISA. As shown in FIG. 4A, the average S/P value of these negative serum samples was 0.095, SD was 0.037, and the threshold for E2-iELISA was determined to be 0.2. To assess the sensitivity of E2-iELISA, the sensitivity of E2-iELISA was assessed using anti-APPV serum (5120) of known NAb titer. The results showed that the E2-iELISA detected serum titres equal to or greater than the corresponding NAb titres, indicating that the E2-iELISA had higher sensitivity (FIG. 4B). The specificity of the E2-iELISA was assessed by detecting the reactivity of porcine antibody positive sera of PRRSV, PCV2, CSFV, PRV and BVDV. As shown in FIG. 4C, no cross-reaction between the expressed recombinant antigen and these sera occurred, indicating that E2-iELISA has good specificity.
In the repeated experiments, 3 APPV antibody positive serum samples and 1APPV antibody negative serum sample were used to determine the intra-and inter-batch CV of the E2-iELISA, at 0.20-5.57% and 4.66-7.07%, respectively (Table 1).
TABLE 1 repeatability of E2-iELISA
Figure BDA0003995079660000121
Figure BDA0003995079660000131
190 sera identified using VNT (including 70 APPV antibody negative sera and 120 APPV antibody positive sera) were used to evaluate E2-iELISA. The E2-iELISA was compared to the VNT to detect the serum samples described above. The E2-iELISA was highly sensitive (113/120, 94.2%) and specific (65/70, 92.9%) to local porcine serum and gave a 93.7% (178/190) response to VNT (Table 2).
TABLE 2 comparison of E2-iELISA with VNT test pig serum samples
Figure BDA0003995079660000132
Test example 2 APPV antibody assay in situ serum samples Using E2-iELISA
In 2017 to 2021, 1368 clinical pig serum samples were collected in 9 provinces in China (Hebei, fujian, sichuan, shanxi, beijing, hubei, heilongjiang, inner Mongolia and Liaoning) and the serum was subjected to retrospective investigation by using an E2-iELISA method established in test example 2, and the test results are shown in tables 3 and 4.
The test results showed that the overall serum prevalence of APPV was 73.9% (1011/1368). The samples are divided according to the sampling areas, and besides the north of the lake, the other cities have the positive rate of 64.04% -86.98%, and the lower positive rate (25%) of the north of the lake is probably due to the lower number of the samples; the samples were divided by year and showed a seropositive rate of 61.63% -89.36% from 2017 to 2021.
TABLE 3 detection of pig farm anti-APPV antibodies by E2-iELISA in different regions of China
Figure BDA0003995079660000133
Figure BDA0003995079660000141
TABLE 4 detection of anti-APPV antibodies in serum of different years in China by E2-iELISA
Figure BDA0003995079660000142
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Claims (10)

1. A method of constructing a mammalian suspension cell line stably expressing APPV E2 protein comprising:
(1) Subcloning the plasmid containing APPV E2 gene into slow virus expression vector to obtain recombinant expression plasmid;
(2) Co-transfecting the recombinant expression plasmid and the auxiliary plasmid into HEK293T cells, and then harvesting recombinant lentivirus;
(3) And (3) transducing the harvested recombinant lentivirus into HEK293 suspension cells to obtain a HEK293-APPV-E2 suspension cell line for stably expressing the APPV E2 protein.
2. The construction method according to claim 1, wherein the APPV E2 gene is optimized according to the synonymous codon bias of mammal, and the nucleotide sequence is shown in SEQ ID No. 1.
3. The construction method according to claim 1, wherein EcoRI restriction sites, kozak sequences and signal peptides are introduced into the N-terminus of the APPV E2 gene in the step (1), and thrombin restriction sites, strep tags, 6 XHis tags and XhoI restriction sites are introduced into the C-terminus.
4. The construction method according to claim 1, wherein in the step (1), a plasmid containing the APPV E2 gene is subcloned into a lentiviral expression vector pLVX-IRES-ZsGreen1 to obtain a recombinant expression plasmid pLVX-APPV-E2.
5. The method of claim 1, wherein in step (2), the recombinant lentivirus is harvested after co-transfection of HEK293T cells with the recombinant plasmid pLVX-APPV-E2 and the helper plasmid pMD2.G and psPAX 2.
6. A mammalian suspension cell line stably expressing APPV E2 protein constructed by the construction method of any one of claims 1-5.
7. A recombinant APPV E2 protein expressed by the mammalian suspension cell line of claim 6.
8. Use of the recombinant APPV E2 protein of claim 7 in the preparation of a reagent for diagnosing or detecting APPV antibodies.
9. An indirect ELISA kit to diagnose or detect APPV antibodies comprising: coating antigen, primary antibody, enzyme-labeled secondary antibody, antibody diluent, washing solution, sealing solution, color development solution and stop solution; wherein the coating antigen is the recombinant APPV E2 protein of claim 7.
10. The indirect ELISA kit according to claim 9, characterized in that it has a detection negative-positive threshold of 0.2.
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* Cited by examiner, † Cited by third party
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CN114015815A (en) * 2021-12-17 2022-02-08 广西壮族自治区动物疫病预防控制中心 Microdroplet digital PCR kit for swine atypical pestivirus and detection method thereof
CN114015815B (en) * 2021-12-17 2024-04-30 广西壮族自治区动物疫病预防控制中心 Microdroplet digital PCR kit for swine atypical pestivirus and detection method thereof

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