CN114773483B - African swine fever virus antigen recombinant tandem epitope protein, construction method and application thereof, and antibody ELISA kit - Google Patents

African swine fever virus antigen recombinant tandem epitope protein, construction method and application thereof, and antibody ELISA kit Download PDF

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CN114773483B
CN114773483B CN202210058058.6A CN202210058058A CN114773483B CN 114773483 B CN114773483 B CN 114773483B CN 202210058058 A CN202210058058 A CN 202210058058A CN 114773483 B CN114773483 B CN 114773483B
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swine fever
fever virus
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virus antigen
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CN114773483A (en
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王乃东
杨文兵
邹亚文
张丽杰
邬静
杨青
王昌建
金业
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Hunan Pujian Biotechnology Co ltd
Hunan Agricultural University
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Hunan Agricultural University
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Abstract

The invention discloses an African swine fever virus antigen recombinant tandem epitope protein, which is formed by connecting an African swine fever virus CD2v protein and a B cell epitope of an MGF360-505R protein in series by using a flexible peptide, wherein the tandem epitope protein has higher expression quantity and inclusion body content, has certain solubility and reactivity with serum antibodies, and can be used for detecting antibody ELISA. Also discloses a construction method of the African swine fever virus antigen recombinant tandem epitope protein, application of the African swine fever virus antigen recombinant tandem epitope protein in antibody ELISA detection and an antibody ELISA detection kit. The application of the invention and the antibody ELISA detection kit have good specificity, good repeatability and stability and high sensitivity, can accurately identify serum samples generated after the ASFV wild type and CD2v gene deletion type strains are immunized, has a certain reference value for ASF detection, and is expected to provide a wider diagnosis range based on a serological detection method of a conserved epitope.

Description

African swine fever virus antigen recombinant tandem epitope protein, construction method and application thereof, and antibody ELISA kit
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an African swine fever virus antigen recombinant tandem epitope protein MGF360-505R-CD2v, a construction method and application thereof, and an antibody ELISA kit.
Background
African swine fever (African swine fever, ASF) is a high-mortality infectious disease in pigs caused by African swine fever virus (African swine fever virus, ASFV). African swine fever virus particles have a structure similar to that of iridovirus, and have the same characteristics as poxviruses in terms of replication, are the only members of the African swine fever virus family, and have been classified into African swine fever virus order (Asfuvirales) and Pokkesviraces (Pokkesviraces) according to the 2019 classification report issued by the International Commission on classification of viruses (International Committee of Taxonomy of Viruses, ICTV).
The related and intensive research of ASF serological diagnosis targets is helpful for the detection of infection, the exploration of pathogenesis, the reaction of the immune system of the organism and the like. The high conservation and antigenicity of important proteins of ASFV (p 30, p72, CD2v, etc.) are of laboratory diagnostic interest. Since ASFV discovery, serological detection methods for different forms of ASFV antibodies have been developed using p30 and p72 awaited selectins.
The ASFV p30 protein is early viral protein (CP 204L gene codes), is an important antigen protein for inducing humoral immune response in the ASFV infection process, and the p30 antibody can prevent ASFV virus from endocytosis, can neutralize virus before or after virus and cell adsorption, and is an important early diagnosis target of virus.
ASFV p72 is a major structural protein expressed in the late stage of infection of ASFV (B646L gene encoding), is a major component of the icosahedral capsid of ASFV, is one of the proteins of high immunogenicity of ASFV, is also an important target for serological diagnosis such as ELISA, and the p72 antibody can prevent adsorption of ASFV to macrophages, but cannot play a decisive role in antibody-mediated immunoprotection;
CD2v is expressed in the late stage of ASFV infection (coded by EP402R gene), is a homolog of CD2, and can cause the phenomenon of virus virulence enhancement and erythrocyte adsorption. The CD2v and C lectin proteins mediate hemagglutination inhibition (Hemagglutination inhibition, HAI) serological specificity of ASFV, and the CD2v and C lectin signature sequences provide a simple method for ASFV serotype grouping. Previous studies considered CD2v less immunogenic, but recent studies found that CD2v protein or its polyclonal antibodies all exhibited good immunoreactivity, suggesting that CD2v protein could be a potential serological diagnostic target.
ASFV MGF genes (e.g., MGF360 and MGF 505) encode Multiple Gene Family (MGF) proteins, and it has been reported that MGF360 and MGF505 genes are involved in inhibiting Interferon (IFN) production; deletion of the MGF gene may reduce replication of ASFV BA71 virus strain in porcine macrophages.
Disclosure of Invention
The invention aims to solve the technical problems and overcome the defects and shortcomings in the background art, and provides an African swine fever virus antigen recombinant tandem epitope protein MGF360-505R-CD2v, a construction method and application thereof, and an antibody ELISA kit.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
an African swine fever virus antigen recombinant tandem epitope protein is formed by connecting an African swine fever virus CD2v protein and a B cell epitope of an MGF360-505R protein in series by using a flexible peptide, namely MGF360-505R-CD2v.
Furthermore, the amino acid sequence of the African swine fever virus antigen recombinant tandem epitope protein (MGF 360-505R-CD2 v) is shown as SEQ ID NO: 1.
Based on a general inventive concept, the invention also provides a construction method of the African swine fever virus antigen recombinant tandem epitope protein, which comprises the following steps: and (3) performing XhoI and XbaI enzyme digestion sites on the encoding genes of the African swine fever virus antigen recombinant tandem epitope protein, subcloning the digested gene fragment into a pET28a vector by using T4 DNA ligase to construct a recombinant plasmid, and then using escherichia coli BL21 to express to obtain the African swine fever virus antigen recombinant tandem epitope protein.
Furthermore, the encoding gene of the African swine fever virus antigen recombinant tandem epitope protein is obtained by designing and screening by the following method: and (3) connecting B cell epitopes of the African swine fever virus p30 protein, the p72 protein, the CD2v protein and the MGF360-505R protein in series by using a python running program, performing automatic permutation and combination, then performing protein solubility prediction on sequences formed by the automatic permutation and combination one by using a protein solubility prediction tool, screening protein sequences with qualified protein solubility prediction values according to requirements, and analyzing to obtain the coding genes of the protein sequences. The program code of the python operating program used therein is shown in the appendix.
Furthermore, the nucleotide sequence of the encoding gene of the African swine fever virus antigen recombinant tandem epitope protein is shown as SEQ ID NO: 2.
Based on a general inventive concept, the invention also provides application of the African swine fever virus antigen recombinant tandem epitope protein in antibody ELISA detection.
Furthermore, the method for carrying out antibody ELISA detection by utilizing the African swine fever virus antigen recombinant tandem epitope protein comprises the following steps:
(1) Diluting the African swine fever virus antigen recombinant tandem epitope protein, and then adding the diluted African swine fever virus antigen recombinant tandem epitope protein into an ELISA plate for coating;
(2) Adding BSA blocking solution into the ELISA plate for blocking incubation;
(3) Diluting a serum sample to be tested, and then adding the diluted serum sample to the ELISA plate for primary antibody incubation;
(4) Diluting HRP-anti-pig lgG, and then adding the diluted HRP-anti-pig lgG into the ELISA plate for enzyme-labeled secondary antibody incubation;
(5) The reaction was quenched with TMB substrate and concentrated sulfuric acid usingSpectrophotometric measurement of OD 450 And (5) numerical value, and judging the result.
Further, in the step (1), the concentration of the African swine fever virus antigen recombinant tandem epitope protein after dilution is 1-6 ng/. Mu.L; in the step (2), the serum sample to be tested is diluted according to the volume ratio of 1:25-200; in the step (3), the enzyme-labeled secondary antibody is diluted according to the volume ratio of 1:5000-8000.
Further, in the step (2), the incubation time of the primary antibody is 30-90min; in the step (5), the time of the color reaction is 5-15min.
Further, in step (5), the result determination is performed according to the following criteria: if the OD of the serum sample to be tested 450 If the value is greater than 0.551, judging positive, and if the OD of the serum sample to be tested is greater than the OD of the serum sample to be tested 450 If the value is less than 0.551, the result is negative.
Based on a general inventive concept, the invention also provides an antibody ELISA detection kit based on the African swine fever virus antigen recombinant tandem epitope protein, which comprises the following components: the antigen-coated 96-well detachable ELISA plate, positive control serum, negative control serum, horseradish peroxidase-labeled goat anti-pig secondary antibody (purchased from KPL company, cat# 14-14-06), 10-time concentrated PBST washing solution, TMB color developing solution (purchased from KPL company, cat# 5120-0076) and stop solution, wherein the antigen is the African swine fever virus antigen recombinant tandem epitope protein.
Further, the positive control serum is immune serum of the african swine fever virus antigen recombinant tandem epitope protein, the negative control serum is pig serum without specific pathogens, the horseradish peroxidase-labeled goat anti-pig secondary antibody is obtained by diluting a stock solution of the goat anti-pig secondary antibody by 1:7000 times of volume, the 10-time concentrated PBST washing solution is phosphate buffer solution with 0.05% Tween 20 and 0.01mol/L, pH value of 7.4, and the stop solution is sulfuric acid solution with 2 mol/L.
Compared with the prior art, the invention has the beneficial effects that:
1. the recombinant tandem epitope protein of the African swine fever virus antigen is formed by connecting B cell epitopes of the African swine fever virus CD2v protein and the MGF360-505R protein in series through flexible peptide, and compared with other tandem epitope proteins such as p30, p72, p30-p72 and the like, the recombinant tandem epitope protein of the African swine fever virus antigen has higher expression level and inclusion body content, has certain solubility and reactivity with serum antibodies, and can be used for detecting antibody ELISA.
2. The construction method of the invention has simple operation, low cost and high synthesis efficiency.
3. The application and antibody ELISA detection kit provided by the invention has the advantages that MGF360-505R-CD2v tandem epitope protein is used for detecting indirect ELISA IgG antibodies, the specificity is good, the repeatability and the stability are good, the sensitivity is high, serum samples generated after ASFV wild type and CD2v gene deletion type strains are immunized can be accurately identified, a certain reference value is provided for ASF detection, and a serologic detection method based on a conserved epitope is expected to provide a wider diagnosis range.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a technical flow chart of the present invention.
FIG. 2 is an ASFV Chinese epidemic strain p30 sequence alignment and its B cell epitope (red portions of the epitope represent amino acids differing from the ASFV Chinese epidemic strain p30 sequence).
FIG. 3 is an ASFV Chinese epidemic strain p72 sequence alignment and its B cell epitope (red portions of the epitope represent amino acids differing from the ASFV Chinese epidemic strain p72 sequence).
FIG. 4 is an ASFV Chinese epidemic strain CD2v sequence alignment and its prediction of B cell epitopes (red portions of epitopes represent amino acids differing from the ASFV Chinese epidemic strain CD2v sequence).
FIG. 5 is a Western Blot His-tag antibody experiment to identify expression of recombinant tandem epitope proteins; wherein M: protein standard Marker; n: a negative control; 1: IPTG non-induced protein expression; 2: IPTG induces protein expression for 1h;3: IPTG induces protein expression for 3h; a: expression of p30 recombinant tandem epitope protein; b: expression of p72 recombinant tandem epitope protein; c: expression of p30-p72 recombinant tandem epitope protein; d: expression of MGF360-505R-CD2v recombinant tandem epitope protein.
FIG. 6 is a soluble analysis of SDS-PAGE to identify recombinant tandem epitope proteins; wherein M: protein standard Marker 1: total protein; 2: supernatant protein after cleavage; 3: precipitating protein after cleavage; a: the solubility analysis of p30 recombinant tandem epitope protein; b: soluble analysis of p72 recombinant tandem epitope protein; c: soluble analysis of p30-p72 recombinant tandem epitope protein; d: soluble analysis of MGF360-505R-CD2v recombinant tandem epitope protein.
FIG. 7 is inclusion body purification of MGF360-505R-CD2v tandem epitope protein; wherein M: protein standard Marker 1: total protein; 2: cleaving the supernatant; 3:2Mol urea treatment of the supernatant; 4:4Mol urea treatment of the supernatant; 5:6Mol urea treatment of the supernatant; 6:8Mol urea treatment of the supernatant.
FIG. 8 is a validation of the reactivity of MGF360-505R-CD2v tandem epitope protein; wherein M: protein standard Marker 1: negative control 2: MGF360-505R-CD2v tandem epitope protein.
FIG. 9 is a standard serum antibody assay; wherein, ASFV Wild type: wild strain serum; ASFV Δcd2v: CD2v gene deletion strain serum.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments and equipment used in the present invention are commercially available or may be prepared by existing methods.
Examples:
an African swine fever virus antigen recombinant tandem epitope protein is formed by tandem connection of a B cell epitope of an African swine fever virus CD2v protein and an MGF360-505R protein by a flexible peptide (namely MGF360-505R-CD2 v), and the amino acid sequence of the protein is shown as SEQ ID NO:1, the nucleotide sequence of the MGF360-505R-CD2v is shown as SEQ ID NO: 2.
The construction and screening process of the african swine fever virus antigen recombinant tandem epitope protein are as follows (shown in figure 1):
1. selection (collection or prediction) of epitopes of African swine fever virus (ASFV Chinese epidemic strain) p30, p72, CD2v and MGF360-505R
The p30, p72 and CD2v sequences of ASFV Chinese epidemic strains (SY 18, HLJ, DB/LN, wbBS01, anhui, CAS19, wuhan1 and Wuhan 2) were downloaded from NCBI (https:// www.ncbi.nlm.nih.gov /), respectively, and ASFV Chinese epidemic strains GenBank accession numbers were: SY18 (MH 766894), HLJ (MK 333180), DB/LN (MK 333181), wbBS01 (MK 645909), anhui (MK 128995), CAS19 (MN 172368), wuhan1 (MN 393476), wuhan2 (MN 393477); and sequence alignment was performed using Weblogo (https:// weblog. Berkeley. Edu/logo. Cgi) to analyze their conservation.
B cell epitopes of ASFV p30, p72 were collected using IEDB (http:// www.iedb.org /). The CD2v proteins were functionally analyzed using InterPro (http:// www.ebi.ac.uk/Interpro /) and HMM method-based protein transmembrane region prediction tools (http:// www.cbs.dtu.dk/services/TMHMM /) and extracellular domains were derived, and B cell epitope predictions were performed on CD2v and MGF360-505R using BepipPred-2.0 (http:// www.cbs.dtu.dk/services/BepipPred /) and IEDB (http:// www.iedb.org /) using different analysis methods.
2. Design of African swine fever Virus p30, p72, CD2v and MGF360-505R antigen tandem epitope proteins isoelectric points (pI, isoelectric point) and molecular weights (Mw, molecular weight) were obtained by means of the Switzerland institute of Bioinformatics (ExpASY proteomics server: http:// ca. ExPASy. Org) using soluble prediction tools for recombinant protein E.coli expression, recombinant Protein Solubility (https:// biotech. Ou. Edu /). And inputting a protein sequence into a Recombinant Protein Solubility forecast tool website page, and clicking and submitting isoelectric points and molecular mass obtained from ExPASy to obtain the solubility forecast.
B cell epitopes of ASFV Chinese epidemic strains p30, p72, CD2v and MGF360-505R are connected in series by flexible peptide (GGGGS) and are arranged and combined, and finally, the solubility analysis is carried out. In order to improve the design efficiency, the P30, P72, CD2v and MGF360-505R B cell epitopes are connected in series by flexible peptide (GGGGS) and are automatically arranged and combined by installing a management program Anaconda, then the existing solubility prediction tool is used for predicting the protein solubility of sequences formed by the automatic arrangement and combination one by one, so that a protein sequence design scheme with high solubility prediction value is obtained, and after the program is operated, all epitopes influencing the protein solubility are removed during the program execution.
Selection of soluble predictive Protein-sol tools for recombinant Protein E.coli expression: (https:// Protein-sol. Mancheter. Ac. Uk /) the website provides a method for rapidly predicting the solubility of E.coli expressed proteins based on sequence, and the Recombinant Protein Solubility prediction results are verified.
3. Expression of antigen tandem epitope proteins of African swine fever virus p30, p72, CD2v and MGF360-505R
The invention relates to a coding gene of p30, p72, p30-p72 and MGF360-505R-CD2v antigen tandem epitope protein which are designed and synthesized by Kirsway biotechnology limited company, wherein after being digested by XhoI and XbaI digestion sites, the digested gene fragments are respectively subcloned into a pET28a vector by using T4 DNA ligase, and respectively named pET28-p30, pET28a-p72, pET28a-p30-p72 and pET28a-MGF360-505R-CD2v recombinant plasmids, the expression strain of an escherichia coli expression system BL21 is used for expression, the Western Blot verification is carried out by using His-tag antibody (CST company), and the solubility analysis is carried out by SDS-PAGE protein gel.
Wherein, the nucleotide sequence of the coding gene of p30 is shown in SEQ ID NO:3, the nucleotide sequence of the coding gene of the p72 is shown as SEQ ID NO: 4), the nucleotide sequence of the coding gene of the p30-p72 is shown as SEQ ID NO:5, the nucleotide sequence of the coding gene of MGF360-505R-CD2v is shown as SEQ ID NO: 2.
Antibody ELISA detection application of MGF360-505R-CD2v antigen tandem epitope protein
4.1 purification and identification of African swine fever Virus CD2v and MGF360-505R antigen tandem epitope protein
Urea purification of the expressed MGF360-505R-CD2v protein inclusion bodies, MGF360-505R-CD2v protein inclusion body purification procedure: (1) Taking MGF360-505R-CD2v 40mL bacterial liquid sediment, adding the lysate, re-suspending inclusion body sediment, and operating under the conditions of an ultrasonic cell grinder of 200W,5s and 5s for 10 min. (2) Centrifuging at 20000xg for 20min at 10 ℃ to obtain inclusion body sediment, adding TritonX-100 to 1% to decompose cell membranes and dissolve membrane proteins, incubating the lysate on ice for 10min, centrifuging at 20000xg for 20min to precipitate inclusion bodies, and finally removing the soluble supernatant to be designated as S. (3) Adding 2M urea concentration buffer solution into the sediment, incubating for 20min, precipitating inclusion bodies at 20000xg for 20min and 10 ℃, removing soluble supernatant to be 2S, treating the inclusion bodies with 4M, 6M and 8M urea according to the step, and respectively taking the supernatants to be 4S, 6S and 8S. (4) SDS-PAGE protein gel is carried out, photographing analysis is carried out, purification of target proteins is determined, and Western Blot is carried out to identify immunogenicity.
4.2 steps of the antibody ELISA detection method
The adopted antibody ELISA detection kit comprises the following components: 96-well removable enzyme-linked reaction plates coated with antigen (antigen is MGF360-505R-CD2v recombinant tandem epitope protein), positive control serum (MGF 360-505R-CD2v recombinant tandem epitope protein immune serum), negative control serum (swine serum without specific pathogen), horseradish peroxidase-labeled goat anti-swine secondary antibody (purchased from KPL company, stock solution diluted 1:7000), 10-fold concentrated PBST wash (0.01 mol/L, pH value 7.4 phosphate buffer containing 0.05% Tween 20), TMB chromogenic solution (purchased from KPL company, stock solution number 5120-0076), and stop solution (2 mol/L sulfuric acid solution).
The antibody ELISA detection method comprises the following specific steps:
(1) Coating: MGF360-505R-CD2v recombinant tandem epitope protein was diluted to the use concentration, incubated at 4℃for 12h at 100. Mu.L per well, and washed 5 times with 300. Mu.L each of PBST (0.05% Tween).
(2) Closing: BSA blocking solution was prepared, 200 μl per well, incubated for 3h at room temperature (24 ℃) and washed 5 times with pbst (0.05% tween).
(3) Primary antibody (serum): after dilution, 100. Mu.L of each well was incubated at room temperature (24 ℃) and washed 5 times with PBST (0.05% Tween).
(4) Enzyme-labeled secondary antibody (HRP-anti-pig lgG): after dilution, 100. Mu.L of each well was incubated at room temperature (24 ℃) and washed 5 times with PBST (0.05% Tween).
(5) Color development and termination: 50. Mu.L of TMB substrate per well, developed at room temperature (24 ℃) and stopped by 50. Mu.L of concentrated sulfuric acid stop solution per well, and the OD was measured by a spectrophotometer 450 And (5) numerical value, and judging the result.
4.3 Determination of optimal coating concentration of MGF360-505R-CD2v protein
MGF360-505R-CD2v protein concentration is diluted to 1, 2, 4 and 6 ng/mu L respectively, 100 mu L of each hole is sequentially longitudinally coated with ELISA plates for experiments, other experimental conditions are kept unchanged, and the optimal coating concentration of MGF360-505R-CD2v protein is judged according to the ratio P/N of the average value of the positive serum and the negative serum compound holes.
4.4 determination of the optimal dilution of serum samples
Serum was mixed at 1: 25. 1: 50. 1: 100. 1: 200. mu.L of each well was added to ELISA plates, duplicate wells were set, and incubated at room temperature (24 ℃). And (3) keeping other experimental conditions unchanged, and judging the optimal dilution of the serum antibody according to the ratio P/N of the average value of the positive serum and the negative serum duplicate wells.
4.5 determination of optimal dilution of the second enzyme-labeled antibody
The enzyme-labeled secondary antibody is prepared according to the following steps of 1: 5000. 1: 6000. 1: 7000. 1:8000 dilution, 100. Mu.L per well was added to ELISA plates, multiplexed wells were set, and incubated at room temperature (24 ℃). And (3) keeping other experimental conditions unchanged, and judging the optimal dilution of the secondary antibody according to the ratio P/N of the average value of the duplicate wells of the positive serum and the negative serum.
4.6 determination of serum optimal response time
The diluted serum is added into ELISA plates, and different reaction time is set: the reaction was carried out for 30min,45min,1h,1.5h, duplicate wells were set, (24 ℃) and incubated at room temperature (24 ℃). And (3) keeping other experimental conditions unchanged, and judging the optimal reaction time of the serum according to the ratio P/N of the average value of the positive serum and the negative serum duplicate wells.
4.7 Determination of TMB time of optimum action
Setting TMB reaction: 5min, 10min, 15min, 50. Mu.L per well, incubation at room temperature (24 ℃). And (3) keeping other experimental conditions unchanged, and judging the optimal acting time of the TMB according to the ratio P/N of the average value of the duplicate wells of the positive serum and the negative serum.
4.8 determination of the Critical value of yin-yang
30 negative control pig serum was tested using an initially established ASFV antibody ELISA method to determine OD 450 Values were calculated and the mean (AV) and Standard Deviation (SD) of 30 negative sera were calculated according to the statistical formula: the mean (AV) +3×standard deviation (SD) is used to calculate the yin-yang limit.
4.9 specificity test
3 parts of positive serum, each of which is collected from a pig farm in Hunan in 2017 and detected as swine fever (CSFV), pseudorabies (PRV), porcine Reproductive and Respiratory Syndrome (PRRSV), porcine circovirus type 2 (PCV 2) and Porcine Epidemic Diarrhea (PEDV), are taken together in 15 parts of serum. The detection was performed using the established antibody ELISA method to set up secondary wells and the average was calculated. And comparing with a critical value to judge the specificity of the method.
4.10 repeatability test
4.10.1 intra-batch differences
Selecting 16 clinical pig serum samples, using the same ELISA plate, performing 3 repetitions of the ELISA method using MGF360-505R-CD2v protein antibody for each serum, and according to OD 450 As a result, an Average Value (AV) and a Standard Deviation (SD) are calculated, and a coefficient of variation is calculated according to a formula: coefficient of variation CV (%) =standard deviation (SD)/Average Value (AV) ×100%.
4.10.2 batch-to-batch differences
16 clinical pig serum samples are selected, ELISA plates are coated in three batches every other day, 3 repeats are carried out on each serum by using an antibody ELISA method of MGF360-505R-CD2v protein, and the ELISA method is carried out according to OD 450 As a result, an Average Value (AV), a Standard Deviation (SD) and a coefficient of variation are calculated according to the formula: coefficient of variation CV (%) =standard deviation (SD)/Average Value (AV) ×100%.
4.11 Preliminary application of ELISA clinical sample detection
2 standard serum samples (ASFV 8-type wild strain and CD2v gene deleted strain) were purchased from chinese veterinary drug administration as standard positive controls; 30 negative control pig serum was collected in a pig farm in the Hunan during 2017; the feasibility and accuracy of the antibody ELISA method for MGF360-505R-CD2v protein are verified by using 99 clinical pig serum samples to detect the antibody ELISA kit for MGF360-505R-CD2v protein in the animal epidemic prevention control center of Hunan province and comparing with the French ID-VET kit.
5. Experimental results
5.1 selection (collection or prediction) of epitopes of the epidemic strains of ASFV in China
Through analysis, the homology of the Chinese ASFV epidemic strain p72 and CD2v is 100%; the homology of the rest sequence area can still reach 100 percent except that p30 lacks the 8 th amino acid (MDFINIS) in the Wuhan1 strain and the Wuhan2 strain, the 194 th amino acid difference (K difference is I) in the SY18 strain and the 195 th to 201 th amino acids (SFFLTYI) are added. Overall, p30, p72 and CD2V protein sequences of chinese epidemic strains are highly conserved.
As a result of comparison analysis of ASFV B cell epitopes reported in IEBD and literature, 5 p30 epitopes (11-30, 24-60, 84-90, 116-125, 146-160 aa) were conserved in Chinese epidemic strains (homology 100%). 13 other epitopes (40-80, 61-84, 61-93, 61-110, 91-103, 91-130, 96-105, 120-204, 123-137, 111-160, 143-182, 175-194, 161-201 aa) have differences in partial amino acids (homology up to 77%) and are shown in FIG. 2;
10 p72 epitopes (37-48, 156-165, 179-210, 242-269, 265-280, 280-294, 290-303, 400-404, 518-552, 586-595 aa) were conserved in chinese pandemic strains (100% homology), and 1 epitope (364-395 aa) was single amino acid different (fig. 3);
according to analysis, the extracellular domain of the CD2v protein is 16-206aa, B cell epitope prediction is carried out on the CD2v and MGF360-505R by using BepiPred-2.0 (http:// www.cbs.dtu.dk/ser devices/BepiPred /) and IEDB (http:// www.iedb.org /) through different analysis options, and unified adjustment is carried out on the predicted B cell epitopes by combining various methods, so that 7 adjusted B cell epitopes are obtained (figure 4); similarly, the B cell epitopes of the MGF360-505R family gene fragment are shown in Table 1.
TABLE 1 prediction of the epitope of the ASFV Chinese epidemic strain MGF360-505R B cell
5.2 design of African swine fever virus antigen tandem epitope protein
After program operation, 8 epitopes are finally selected and connected by using flexible peptide to form the ASFV p30 recombinant tandem epitope protein, the solubility prediction result of the Previous Model (1991) is 100%, and when the solubility result of the prediction tool is more than 75%, the recombinant protein can be expressed in a soluble way without fusing other fusion promotion tags (or fusion promotion molecular chaperones), and the comprehensive accuracy is 86%. With the Protein-sol solubility prediction tool as a further validation (the method was fully divided into 1 and had solubility at greater than 0.45), the prediction was 0.846. The predicted results of Previous Model (1991) and Protein-sol (Table 2) were in agreement, and in subsequent applications, the same Protein had multiple epitopes in close proximity to each other, so that the amino acids spaced between epitopes were not deleted to ensure that antigenicity was not affected, and remained directly intact.
After program operation, the ASFV p72 recombinant tandem epitope Protein finally selects 8 epitopes, and is formed by connecting flexible peptides, the solubility prediction result of a Previous Model (1991) is 100%, and the Protein-sol verification prediction result is 0.565,Previous Model (1991) and the Protein-sol prediction result accords with (Table 2).
After program operation, the ASFV p30-p72 recombinant tandem epitope Protein is formed by finally selecting 12 epitopes and connecting the epitopes through flexible peptides, the solubility prediction result of a Previous Model (1991) is 100%, the Protein-sol verification prediction result is 0.773, and the prediction result accords with (table 3).
After program operation, ASFV MGF360-505R-CD2v recombinant tandem epitope Protein is formed by finally selecting 19 epitopes and connecting the epitopes through flexible peptide, the solubility predicted result of a Previous Model (1991) is 99.9%, and the Protein-sol verification predicted result is 0.775, wherein the predicted result accords with (table 4).
TABLE 2 epitope sequence and solubility prediction of ASFV p30, p72 recombinant tandem epitope proteins
TABLE 3 epitope sequence and solubility prediction of ASFV p30-p72 recombinant tandem epitope protein
TABLE 4 epitope sequence and solubility prediction of ASFV MGF360-505R-CD2v recombinant tandem epitope protein
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5.3 preparation of African swine fever Virus antigen tandem epitope protein
5.3.1 expression of African swine fever Virus antigen tandem epitope protein
The expression of the antigen tandem epitope protein is verified by using His-tag antibody in Western Blot, and the result shows that the antigen tandem epitope protein is expressed, wherein the expression quantity of p30 and MGF360-505R-CD2v tandem epitope proteins is higher, the expression quantity of p30-p72 tandem epitope protein is good, and the expression quantity of p72 tandem epitope protein is lower (figure 5).
5.3.2 soluble analysis of African swine fever Virus antigen tandem epitope protein
SDS-PAGE results show that p30, p30-p72, MGF360-505R-CD2v recombinant tandem epitope proteins all have a certain solubility (FIG. 6).
5.3.3 Purification of MGF360-505R-CD2v tandem epitope protein
Prokaryotic expression MGF360-505R-CD2v tandem epitope protein has high expression quantity and inclusion body content, and the protein can be used for distinguishing wild type strains from antibodies generated by MGF360-505R-CD2v gene deletion strains in early design and later application. Therefore, we selected MGF360-505R-CD2v tandem epitope protein inclusion bodies for urea purification to give purer target protein (fig. 7).
5.3.4 Reactivity verification of MGF360-505R-CD2v tandem epitope protein
The Western Blot serum reactivity experiment verifies that the MGF360-505R-CD2v tandem epitope protein reacts with the pig positive serum antibody, and the result shows that the MGF360-505R-CD2v tandem epitope protein has reactivity with the pig positive serum antibody in the Western Blot experiment (FIG. 8).
5.4 Establishment of antibody ELISA of MGF360-505R-CD2v epitope tandem protein
5.4.1 determination of protein coating concentration
The concentration of the coated antigen protein was optimized, the OD value results are shown in Table 5, and the P/N value was calculated. Analysis of the results showed that the P/N ratio was highest at a concentration of coated antigen of 4 ng/. Mu.L (8.3553).
TABLE 5 determination of optimal coating concentration of proteins
5.4.2 determination of the optimal dilution of serum samples
Serum dilution was changed, other conditions were kept unchanged, the results of the OD values were shown in Table 6, and the P/N values were calculated. Analysis of the results showed that the P/N was higher when the weakly positive serum was diluted with 100-fold serum antibody (4.0919).
TABLE 6 determination of optimal dilutions of serum antibodies
5.4.3 determination of optimal dilution of the second enzyme-labeled antibody
The secondary antibody dilution was changed, other conditions were kept unchanged, the OD value was measured as shown in Table 7, and the P/N value was calculated. Analysis of the results showed that the P/N was highest at a dilution of 1:7000 for the secondary antibody (7.6067).
TABLE 7 determination of optimal dilution of enzyme-labeled antibodies
5.4.4 Determination of TMB time of optimum action
The TMB time was varied, other conditions were maintained, the OD values were measured as shown in Table 8, and the P/N values were calculated. Analysis of the results showed that TMB was high in P/N at 5min (8.2675).
TABLE 8 determination of optimal color development time
5.4.5 determination of serum optimal response time
The serum reaction time was changed, other conditions were kept unchanged, the OD value was measured as shown in Table 9, and the P/N value was calculated. Analysis of the results showed that the P/N was highest at 30min of primary antibody action (12.6273).
TABLE 9 determination of optimal reaction time for serum antibodies
5.4.6 determination of the critical value
30 negative pig serum samples collected in a pig farm in Hunan in 2017 were selected and detected by an established antibody ELISA method, and the results are shown in Table 10, wherein the average OD of 30 ASFV negative serum 450 The value (AV) is 0.224 and the Standard Deviation (SD) is 0.109, according to the formula: the Average Value (AV) +standard deviation (SD) ×3 calculated threshold value is 0.551. Detecting sample OD 450 If the value is greater than 0.551, the result is positive, and if the value is less than 0.551, the result is negative.
Determination of critical values of Table 10
Sample number OD 450 Sample number OD 450 Sample number OD 450
1 0.0588 11 0.2713 21 0.4623
2 0.1017 12 0.2373 22 0.1832
3 0.1460 13 0.3736 23 0.3765
4 0.1321 14 0.0703 24 0.3121
5 0.1603 15 0.2925 25 0.1330
6 0.3099 16 0.2176 26 0.4406
7 0.2284 17 0.2126 27 0.3699
8 0.0801 18 0.2722 28 0.1299
9 0.1692 19 0.1360 29 0.1122
10 0.1457 20 0.2966 30 0.2943
5.4.7 specificity test
Swine Fever (CSFV), porcine Pseudorabies (PRV), porcine Reproductive and Respiratory Syndrome (PRRSV), porcine circovirus type 2 (PCV 2), porcine Epidemic Diarrhea (PEDV) positive serum was 15 parts total, tested using the established antibody ELISA method, and negative and positive controls were set up, the results are shown in Table 11, swine fever (CSFV), porcine Pseudorabies (PRV), porcine Reproductive and Respiratory Syndrome (PRRSV), porcine circovirus type 2 (PCV 2), porcine Epidemic Diarrhea (PEDV) positive serum OD 450 The average value is smaller than the threshold value of 0.551, and the detection is negative, which indicates that the established ELISA method has good specificity.
TABLE 11 results of indirect ELISA specificity experiments
5.4.8 repeatability test
5.4.8.1 in-batch repeat test
16 samples of pig serum, 3 replicates per serum, were obtained in the microplate reader as shown in table 12, the mean (AV), standard Deviation (SD) were calculated, and the coefficient of variation was calculated according to the formula: coefficient of variation CV (%) = Standard Deviation (SD)/Average Value (AV) ×100%, resulting in an inter-lot coefficient of variation of 0.9611% -6.6319%, less than 10%, indicating better inter-lot reproducibility and stability of the established antibody ELISA method.
TABLE 12 Indirect ELISA in-batch repeat assay
5.4.8.2 repeated test between batches
16 pig serum samples were selected, three ELISA plates were repeated 3 times for each serum, the results obtained in the microplate reader are shown in Table 13, the Average Value (AV), standard Deviation (SD) were calculated, and the coefficient of variation was calculated according to the formula: coefficient of variation CV (%) = Standard Deviation (SD)/Average Value (AV) ×100%, resulting in an inter-lot coefficient of variation of 0.8723% -7.7009%, less than 10%, indicating that the established antibody ELISA method has better inter-lot reproducibility and stability.
TABLE 13 indirect ELISA batch-to-batch repeat test
5.4.9 Sample detection for ELISA
The standard serum verification result is shown in fig. 9, and can accurately identify serum samples generated after the wild type ASFV and the CD2v gene deletion type strain are immunized, wherein the coincidence rate of MGF360-505R-CD2v prokaryotic expression tandem epitope protein and French ID-VET detection kit is 92.93 percent (92/99), and the positive coincidence rate is: 82.6% (19/23), negative compliance rate: 96.05% (73/76).
In conclusion, the African swine fever virus antigen recombinant tandem epitope protein is formed by connecting the African swine fever virus CD2v protein and the B cell epitope of the MGF360-505R protein in a flexible peptide manner, and compared with other tandem epitope proteins such as p30, p72, p30-p72 and the like, the MGF360-505R-CD2v tandem epitope protein has higher expression quantity and inclusion body content, certain solubility and reactivity with serum antibodies, and can be used for detecting antibody ELISA.
Appendix:
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Sequence listing
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<120> African swine fever virus antigen recombinant tandem epitope protein, construction method and application thereof, and antibody ELISA kit
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Asp Asn Gly Ser Gly Gly Gly Pro Ile Leu Glu Asn Lys Phe Val Gly
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ctgaaaccgc gtgaagagta ccagccgtcc ggcggtggtg gctcgcaaaa ggacctggtt 900
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ttcctgagcc agcattttgg tggtggtggc agccatcacc accaccacca ctaa 1194

Claims (6)

1. The african swine fever virus antigen recombinant tandem epitope protein is characterized in that the antigen recombinant tandem epitope protein is formed by tandem connection of a B cell epitope of an african swine fever virus CD2v protein and an MGF360-505R protein by a flexible peptide, and the amino acid sequence of the antigen recombinant tandem epitope protein is shown as SEQ ID NO: 1.
2. A method for constructing the african swine fever virus antigen recombinant tandem epitope protein according to claim 1, comprising the steps of: and (3) performing XhoI and XbaI enzyme digestion sites on the encoding genes of the African swine fever virus antigen recombinant tandem epitope protein, subcloning the digested gene fragment into a pET28a vector by using T4 DNA ligase to construct a recombinant plasmid, and expressing by using escherichia coli BL21 to obtain the African swine fever virus antigen recombinant tandem epitope protein.
3. The construction method according to claim 2, wherein the encoding gene of the african swine fever virus antigen recombinant tandem epitope protein is designed and screened by the following method: and (3) connecting B cell epitopes of the African swine fever virus p30 protein, the p72 protein, the CD2v protein and the MGF360-505R protein in series by using a python running program, performing automatic permutation and combination, then performing protein solubility prediction on sequences formed by the automatic permutation and combination one by using a protein solubility prediction tool, screening protein sequences with qualified protein solubility predicted values according to requirements, and analyzing to obtain the coding genes of the protein sequences.
4. The construction method according to claim 2, wherein the nucleotide sequence of the encoding gene of the african swine fever virus antigen recombinant tandem epitope protein is as shown in SEQ ID NO: 2.
5. An antibody ELISA detection kit based on the african swine fever virus antigen recombinant tandem epitope protein of claim 1 or obtained by the construction method of any one of claims 2 to 4, comprising: the kit comprises a 96-well detachable enzyme-linked reaction plate coated with antigen, positive control serum, negative control serum, horseradish peroxidase-labeled goat anti-pig secondary antibody, PBST washing liquid, TMB color development liquid and termination liquid.
6. The antibody ELISA detection kit according to claim 5, wherein the positive control serum is immune serum of the african swine fever virus antigen recombinant tandem epitope protein, the negative control serum is pig serum without specific pathogens, the horseradish peroxidase-labeled goat anti-pig secondary antibody is obtained by diluting a stock solution thereof by 1:7000 times of volume, the PBST washing solution is phosphate buffer solution with 0.01mol/L, pH value of 7.4 containing 0.05% Tween 20, and the stop solution is sulfuric acid solution with 2 mol/L.
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