CN116023506B - ASFV nonstructural protein dominant antigen epitope fusion protein, kit and application thereof - Google Patents
ASFV nonstructural protein dominant antigen epitope fusion protein, kit and application thereof Download PDFInfo
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Abstract
The invention provides an ASFV nonstructural protein dominant antigen epitope fusion protein, a kit and application thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the ASFV non-structural protein dominant antigen epitope fusion protein is shown as SEQ ID No. 1. The fusion protein has good immunoreactivity with the African swine fever virus positive serum. An ELISA detection method for detecting the African swine fever virus nonstructural protein antibody is established by using the obtained ASFV nonstructural protein dominant antigen epitope fusion protein as a coating antigen; the detection kit has the characteristics of strong specificity, high sensitivity, good repeatability and high accuracy, can effectively detect the antibody condition generated after African swine fever virus infection, and provides important help for diagnosis of African swine fever, research on infection mechanism and development of safe and efficient vaccines.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an ASFV nonstructural protein dominant antigen epitope fusion protein, a kit and application thereof.
Background
African swine fever (African Swine fever, ASF) is a highly contagious disease of pigs caused by African swine fever virus (African Swine fever virus, ASFV), and has clinical symptoms and pathological changes very similar to those of classical swine fever, and higher morbidity and mortality. ASFV is a linear double-stranded DNA virus, mature virions are 20-sided symmetrical and have a envelope, the size of the genome varies between strains to a certain extent, generally between 170 and 190kb, the genome contains 150 open reading frames, approximately encoding more than 100 nonstructural proteins and 68 structural proteins. The nonstructural proteins play an important role in viral replication and assembly, host cell function regulation, host natural immunity escape and the like. However, the detection methods of the related ASFV nonstructural protein antibodies are quite few, and most of the detection methods are aimed at the detection of the African swine fever virus structural protein antibodies in clinical application, so that the deep research and understanding of the related pathogenic mechanism of the African swine fever virus are severely restricted.
The A238L protein is an important non-structural protein of the African swine fever virus, can regulate related biological functions of host cells in the infection process of the virus, and creates a good living space for the African swine fever virus. Since this protein contains 1kb ankyrin repeat sequence, it is considered as a 1kb analogue. In different transfected cells, the protein has slightly different molecular weights due to the influence of different intracellular environments. Studies of Silk et al show that A238L protein can exist in cytoplasm and nucleus simultaneously, and accumulate in nucleus 10-18 h after infection, can inhibit activation of NF-kappaB, and assist natural immune response of African swine fever virus escaping host. In addition, the A238L protein can inhibit the activation of transcription factors related to activated T cell nuclear factors, so that the related immune function of host cells is lost, and the replication and transcription of African swine fever virus particles are promoted. The A238L protein can inhibit transcription of inducible nitric oxide synthase genes by inhibiting p65 acetylation and p300 activity in macrophages, thereby reducing the generation of inflammatory mediators, nitric oxide, and playing an anti-inflammatory role. However, in the research process of the related action mechanism of the A238L protein molecule, prokaryotic expression and eukaryotic expression are mostly carried out on the whole gene sequence of the African swine fever virus nonstructural protein A238L, so that the expressed sequence contains a sequence which has no interaction with host protein, the interaction between the A238L protein molecule and the target molecule sequence is seriously influenced, and the interaction site between the A238L protein molecule and the target protein molecule cannot be accurately displayed; in addition, the inclusion of a non-functional sequence may affect the expression level of the recombinant protein of interest or cause the recombinant protein of interest not to be expressed, etc. The technology of the item carries out tandem expression on the dominant antigen epitope of African swine fever virus African structural protein A238L, eliminates the sequence without related action, can fully present the dominant antigen epitope of the protein, improves the binding efficiency of the protein and an African swine fever virus antibody, can accurately detect the African swine fever virus African structural protein antibody as a coating antigen so as to judge the epidemic dynamics of the African swine fever virus in a pig group and the infection replication rule of the African swine fever virus in the pig body, and provides important help for diagnosis of African swine fever, research of infection mechanism and development of safe and efficient vaccines.
Disclosure of Invention
Therefore, the invention aims to provide an African swine fever virus non-structural protein dominant antigen epitope fusion protein, a kit and application thereof, wherein 4 dominant antigen epitopes of an ASFV non-structural protein A238L protein are connected in series to obtain the ASFV non-structural protein dominant antigen epitope fusion protein, and the fusion protein has good immunoreactivity with an African swine fever virus positive serum. The kit uses ASFV nonstructural protein dominant antigen epitope fusion protein as a coating antigen, and has the characteristics of good specificity, sensitivity, repeatability and accuracy when detecting whether an ASFV antibody is contained in pig serum.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an African swine fever virus non-structural protein dominant antigen epitope fusion protein, and the amino acid sequence of the African swine fever virus non-structural protein dominant antigen epitope fusion protein is shown as SEQ ID No. 1.
The invention provides a nucleotide for encoding the african swine fever virus nonstructural protein dominant antigen epitope fusion protein.
Preferably, the nucleotide sequence is shown as SEQ ID No. 2.
The invention provides an expression vector containing the nucleotide sequence.
The invention provides a host bacterium containing the nucleotide sequence or the expression vector.
The invention provides an ELISA antibody detection kit for an African swine fever virus nonstructural protein dominant antigen epitope fusion protein, which comprises an ELISA plate, wherein the ELISA plate is coated with the African swine fever virus nonstructural protein dominant antigen epitope fusion protein.
Preferably, the kit further comprises a coating liquid, a washing liquid and a sealing liquid.
The invention provides a preparation method of an African swine fever virus nonstructural protein dominant antigen epitope fusion protein, which comprises the following steps:
constructing the expression vector, then converting the expression vector into host bacteria, performing ultrasonic disruption, inclusion body dissolution and purification after inducing expression on host cells containing the African swine fever virus non-structural protein dominant antigen epitope fusion protein, and collecting to obtain the African swine fever virus non-structural protein dominant antigen epitope fusion protein.
The invention provides a preparation method of an African swine fever virus nonstructural protein antibody, which comprises the following steps:
the fusion protein is used as antigen to immunize animal and produce african swine fever virus non-structural protein antibody in animal body.
The invention provides application of the fusion protein, nucleotide, expression vector or host bacterium in preparation of African swine fever virus products.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an African swine fever virus nonstructural protein dominant antigen epitope fusion protein, a kit and application thereof, wherein 4 dominant antigen epitopes of an ASFV nonstructural protein A238L protein are connected in series to obtain the ASFV nonstructural protein dominant antigen epitope fusion protein, and the fusion protein has good immunoreactivity with African swine fever virus positive serum. The kit utilizes ASFV nonstructural protein dominant antigen epitope fusion protein as a coating antigen, and the established antibody detection kit has the characteristics of strong specificity, high sensitivity, good repeatability and high accuracy, can effectively detect antibodies generated after African swine fever virus infection so as to judge epidemic dynamics of the African swine fever virus in a pig group and infection replication rules of the African swine fever virus in the pig body, and provides important help for diagnosis of African swine fever, research of infection mechanisms and development of safe and efficient vaccines.
Drawings
FIG. 1 shows amplification of 4 dominant antigen epitope tandem fusion proteins of African swine fever virus nonstructural protein A238L, carrier construction and identification of expression strain construction, wherein M is DL2000 DNA Mark, lane 1 is the result of PCR amplification of A238L-B gene sequence, lane 2 is the result of construction of a Pet28a-A238L-B expression carrier, and lane 3 is the result of identification of E.Coli BL21/Pet28a-A238L-B bacterial liquid.
FIG. 2 shows SDS-PAGE electrophoresis identification of 4 dominant epitope fusion proteins of African swine fever virus nonstructural protein A238L after induced expression purification, wherein M: protein markers; lane 1 is 4 dominant epitope fusion proteins expressing purified african swine fever virus nonstructural protein a 238L.
FIG. 3 is an identification of the reactivities of 4 dominant epitope fusion proteins of African swine fever virus nonstructural protein A238L, wherein M: protein markers; lane 1 is 4 dominant epitope fusion proteins expressing purified african swine fever virus nonstructural protein a 238L.
Detailed Description
In order to fully present the dominant epitope of the African swine fever virus nonstructural protein A238L protein and improve the specificity, sensitivity and accuracy of the protein antigen in detection, a high-titer antibody of the protein is prepared, the whole gene sequence of the African swine fever virus nonstructural protein A238L protein is analyzed, 4 dominant epitopes in the A238L protein are found to have better immunoreactivity, and the 4 dominant epitope gene sequences of the African swine fever virus nonstructural protein A238L protein are synthesized in series in African swine fever virus with different genotypes, so that the invention provides the African swine fever virus nonstructural protein dominant epitope fusion protein, and the amino acid sequence of the African swine fever virus nonstructural protein dominant epitope fusion protein is shown as SEQ ID No. 1.
The invention also provides a nucleotide for encoding the african swine fever virus nonstructural protein dominant antigen epitope fusion protein. The nucleotide sequence is preferably shown as SEQ ID No. 2.
The invention also provides an expression vector containing the nucleotide. In the present invention, the basic vector of the expression vector is preferably a Pet28a vector.
The invention provides a host bacterium containing the nucleotide or the expression vector. In the present invention, the starting strain of the host bacterium is preferably E.coli BL21 (DE 3), and the source of E.coli BL21 (DE 3) is not particularly limited, and a commercially available product in the art may be used.
The invention provides an ELISA antibody detection kit for an antigen epitope fusion protein of an African swine fever virus nonstructural protein, which comprises an ELISA plate, wherein the ELISA plate is coated with the antigen epitope fusion protein of the African swine fever virus nonstructural protein.
In the present invention, the kit further preferably includes a coating liquid, a washing liquid, and a blocking liquid. The coating liquid is preferably 0.03-0.06 mol/L carbonate buffer solution, and the pH value is preferably 9.0-10.0. The washing solution is PBST solution, and the PBST solution is a conventional reagent, and the source of the PBST solution is not particularly limited, and the PBST solution can be prepared by self or purchased. The blocking solution is preferably BSA in an amount of 0.005 to 0.015 g/mL. In the invention, the result judgment standard of the kit detection method is as follows: when negative control OD 450nm Average value (N) less than 0.25, positive control OD 450nm When the average value (P) is greater than 0.65, the OD of the sample is 450nm Value (S)/negative control OD 450nm When the average value (N) is more than or equal to 2.1, the sample OD is positive 450nm Value (S)/negative control OD 450nm When the average value (N) is less than 2.1, the test is negative.
The kit has the characteristics of strong specificity, high sensitivity, good repeatability and high accuracy, and can effectively detect antibodies generated after African swine fever virus infection so as to judge whether the African swine fever virus is infected in pigs.
The invention provides a preparation method of an African swine fever virus nonstructural protein dominant antigen epitope fusion protein, which comprises the following steps:
constructing the expression vector, then converting the expression vector into host bacteria, performing ultrasonic disruption, inclusion body dissolution and purification after inducing expression on host cells containing the African swine fever virus non-structural protein dominant antigen epitope fusion protein, and collecting to obtain the African swine fever virus non-structural protein dominant antigen epitope fusion protein.
In the invention, the expression vector is constructed, the basic vector of the expression vector is preferably a Pet28a vector, and the nucleotide of the dominant antigen epitope fusion protein for encoding the african swine fever virus nonstructural protein is named as an A238L-B gene sequence and is connected between BamH L and Xho L restriction enzyme cleavage sites in the Pet28a vector to obtain the Pet28a-A238L-B.
In the invention, after the expression vector is constructed, the expression vector is transformed into host bacteria, the transformation mode is preferably heat stress transformation, and the initial strain of the host bacteria is preferably escherichia coli BL21 (DE 3) to obtain E.Coli BL21/pET28a-A238L-B.
In the invention, after inducing and expressing host cells containing the African swine fever virus nonstructural protein dominant antigen epitope fusion protein, performing ultrasonic crushing, inclusion body dissolution and purification, and collecting to obtain the African swine fever virus nonstructural protein dominant antigen epitope fusion protein. Preferably, the induced expression is induced by IPTG with a final concentration of 0.5-1.5 mmol/L. The ultrasonic crushing condition is preferably 350-450W power, the ultrasonic crushing time is 3-7 s, the interval time is 4-6 s, and the ultrasonic is 25-35 min. The purification preferably comprises His-Bind resin and dialysis, and when His-Bind resin is purified, rinsing liquid C and rinsing liquid D are used for washing and eluting, wherein the rinsing liquid C is preferably 7-9 mol/L urea and 0.05-0.15 mol/L NaH 2 PO 4 0.005-0.015 mol/L Tris-cl pH6.3; the rinsing liquid D is preferably 7-9 mol/L urea and 0.05-0.15 mol/L NaH 2 PO 4 0.005-0.015 mol/L Tris-cl pH5.9; the eluent is preferably 7 to 9mol/L urea and 0.05 to 0.15mol/L NaH 2 PO 4 0.005-0.015 mol/L Tris-cl pH4.5. The purity of the African swine fever virus nonstructural protein dominant antigen epitope fusion protein prepared by the method is up to 95%, and the African swine fever positive serum has good immunoreactivity.
The invention provides a preparation method of an African swine fever virus nonstructural protein antibody, which comprises the following steps:
the fusion protein is used as antigen to immunize animal and produce african swine fever virus non-structural protein antibody in animal body. The antibody of the African swine fever virus nonstructural protein is high in titer, and the titer of the rabbit serum antibody produced by the fusion protein is 1:128 000.
The invention provides application of the fusion protein, nucleotide, expression vector or host bacterium in preparation of African swine fever virus products. In the present invention, the product includes a kit or a reagent.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 preparation of African swine fever Virus nonstructural protein dominant epitope fusion protein
(1) Screening and synthesis of gene sequence of african swine fever virus nonstructural protein dominant antigen epitope fusion protein
In the embodiment, the bioinformatics method is adopted to comprehensively analyze the hydrophilicity, antigenicity and amino acid biological surface accessibility of the secondary structure of African swine fever isolate Pig/HLJ/2018 (MK 333180) A238L protein in GenBank, 4 dominant antigen epitope peptide fragments are screened, namely, the 15 th to 46 th amino acid sequences (N ' -IKKHIRNGNLTLFEEFFKTDPWIVNRCDKNGS-C ', SEQ ID No. 3) of the N ' end of the A238L protein, the 36 th amino acid sequences (N ' -FEQESYPGEIINPHRRDKDGNSALHYLAEKKNHLIL-C ', SEQ ID No. 4) of the 65 th to 100 th sections, the 16 th amino acid sequences (N ' -GADPTQKDYHRGFTAW-C ', SEQ ID No. 5) of the 144 th to 159 th sections and the 9 th amino acid sequences (N ' -TGGLRKSPK-C ', SEQ ID No. 6) of the 195 th to 203 th sections. The gene sequences corresponding to the 32 amino acids of the 15 th to 46 th sections of the N ' end are (5'-attaaaaaacatattagaaatgggaatcttacactatttgaggaattttttaaaacagatccgtggattgtcaatagatgcgataaaaatggatcc-3', 96bp, SEQ ID No. 7), the gene sequences corresponding to the 36 amino acids of the 65 th to 100 th sections are (5'-tttgaacaagaatcttatcctggagaaataattaaccctcataggagggataaagatggaaactctgctttacattatttagctgagaaaaaaaatcatttaatcctg-3', 108bp,SEQ ID No.8), the gene sequences corresponding to the 16 amino acids of the 144 th to 159 th sections are (5'-ggagcagatccgactcaaaaagactatcatagaggttttactgcttgg-3', 48bp, SEQ ID No. 9), and the gene sequences corresponding to the 9 amino acids of the 195 th to 203 th sections are (5'-accggtggtttaagaaaaagcccaaaa-3', 27bp,SEQ ID No.10). The gene sequences corresponding to the amino acid sequences of the 4 dominant epitopes are sent to Shanghai bioengineering limited company for serial synthesis, and the African swine fever virus nonstructural protein dominant epitope fusion protein is obtained.
The amino acid sequence of the african swine fever virus nonstructural protein dominant antigen epitope fusion protein is as follows (SEQ ID No. 1):
n '-IKKHIRNGNLTLFEEFFKTDPWIVNRCDKNGSFEQESYPGEIINPHRRDKDGNSALHYLAEKKNHLILGADPTQKDYHRGFTAWTGGLRKSPK-C',93 amino acid sequences.
The nucleotide sequence of the African swine fever virus epitope fusion protein is as follows (SEQ ID No. 2):
5’-attaaaaaacatattagaaatgggaatcttacactatttgaggaattttttaaaacagatccgtggattgtcaatagatgcgataaaaatggatcctttgaacaagaatcttatcctggagaaataattaaccctcataggagggataaagatggaaactctgctttacattatttagctgagaaaaaaaatcatttaatcctgggagcagatccgactcaaaaagactatcatagaggttttactgcttggaccggtggtttaagaaaaagcccaaaa-3’，279bp。
(2) Construction of African swine fever virus nonstructural protein dominant antigen epitope fusion protein expression vector
According to the gene sequence of the african swine fever virus nonstructural protein dominant antigen epitope fusion protein synthesized by the screening, which is named as A238L-B gene sequence, primers A238L-B-F and A238L-B-R are designed and sent to Shanghai bioengineering limited company for synthesis. Wherein primer A238L-B-F has an EcoRI restriction site (underlined) and a protective base added; the primer A238L-B-R is added with XhoI restriction enzyme cutting site (underlined) and protective base, and PCR amplification is carried out by taking the synthesized A238L-B gene sequence (279 bp) as a template.
A238L-BF：5’-cggaattcattaaaaaacatattagaaatg-3’(SEQ ID No.11)；
A238L-BR：5’-ggctcgagttttgggctttttcttaaac-3’(SEQ ID No.12)。
The PCR amplification system for the A238L-B gene sequence was 50. Mu.L: 2. Mu.L of 5 XPimeSTAR Buffer 10. Mu.L, 100. Mu. Mol/L A L-BF/R primer each 1. Mu.L, 2.5mmol/L dNTP mix 4. Mu.L, 2.5U/. Mu. L PrimeSTAR HS DNA Polymerase 0.5.0.5. Mu.L, ddH of the synthesized template 2 O31.5. Mu.L. The PCR reaction conditions were: after denaturation at 95 ℃ for 5min, the mixture enters a cycle, the cycle parameters are 95 ℃ 50s,52 ℃ 20s,72 ℃ 30s, and after 30 cycles, the mixture is extended for 10min at 72 ℃. After the completion, the specific target fragment of the A238L-B gene sequence was purified and recovered by agarose gel identification at a concentration of 1% (see FIG. 1).
The A238L-B gene sequence recovered by the purification was digested with EcoRI and XhoI, and simultaneously plasmid vector Pet28a was digested with EcoRI and XhoI, and then the A238L-B sequence fragment was ligated with plasmid vector Pet28 a. The system is as follows: A238L-B sequence (EcoRI-XhoI cleavage) 4. Mu.L, pet28a (EcoRI-XhoI cleavage) 4. Mu.L, 10 Xligation buffer 1. Mu.L, T4 DNA ligase 1. Mu.L, ligation at 4℃for 16 hours, and then heat-stress transformation into BL21 (DE 3) competence were performed, and the resultant was plated on LB plates supplemented with kanamycin at a final concentration of 50. Mu.g/mL, incubated at 37℃for 16 hours, and single colonies were picked up and inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, and plasmids were extracted for identification. PCR identification screening BL21 (DE 3) strain BL21 (DE 3) containing plasmid Pet28a-A238L-B (E.Coli BL21/Pet28 a-A238L-B) (see FIG. 1) was obtained.
(3) Expression, purification and reactogenicity of african swine fever virus non-structural protein dominant antigen epitope fusion protein
After picking E.Coli BL21/pET28a-A238L-B single colonies, overnight culture, transfer to freshly prepared LB liquid medium containing kanamycin (50. Mu.g/mL) at 1% inoculum size, shake culture at 37℃at 240rpm for 2h, OD 600nm IPTG was added to a final concentration of 1.0mmol/L to 0.8-1.0, the mixture was transferred to 37℃and cultured with shaking at 240rpm for 6 hours, and the cells were collected by centrifugation. Suspending the obtained strain in PBS, adding lysozyme with final concentration of 1mg/mL, standing on ice for 30min, performing ultrasonic lysis to break cells (400W power, ultrasonic breaking time 5s, interval time 5s, ultrasonic 30 min), centrifuging at 4deg.C at 10000rpm for 15min, and discarding supernatant to obtain inclusion body precipitate. The inclusion bodies were then dissolved in binding buffer (8 mol/L urea, 0.1mol/L NaH) 2 PO 4 0.01mol/L Tris-CL, pH 8.0), and after inclusion body dissolution, centrifuging at 10000rpm at 4℃for 15min, collecting supernatant, pre-equilibrating with His-Bind resin and binding buffer for 1h, discarding filtrate, and washing with rinse C (8 mol/L urea; 0.1mol/L NaH 2 PO 4 The method comprises the steps of carrying out a first treatment on the surface of the Washing and filtering twice with 0.01mol/L Tris-CL pH6.3, and then washing liquid D (8 mol/L urea; 0.1mol/L NaH 2 PO 4 The method comprises the steps of carrying out a first treatment on the surface of the Washing and filtering for 4 times with 0.01mol/L Tris-CL pH5.9, and finally collecting target protein (8 mol/L urea; 0.1mol/L NaH 2 PO 4 ；0.01mol/L Tris-CL ph 4.5). The recombinant protein purified by His-Bind resin is put into a semi-permeable membrane (boiled in water at 100 ℃ for 10 min) which is treated in advance, and reduced glutathione with the final concentration of 1mg/mL is added into the semi-permeable membrane, and the recombinant protein is slowly dialyzed in PBS (phosphate buffer solution) containing 4mol/L, 3mol/L, 2mol/L and 1mol/L urea and pH of 8.5 respectively at the temperature of 4 ℃, and purified A238L-B protein is collected after dialysis, the protein concentration is measured, and SDS-PAGE electrophoresis identification is carried out.
As a result, as shown in FIG. 2 below, a specific African swine fever virus non-structural protein dominant epitope fusion protein band (about 33.0 kd) was successfully obtained, and the purity of the African swine fever virus epitope fusion protein was 95%.
After SDS-PAGE electrophoresis, western-Blot was performed to identify the immunogenicity of the protein, and nitrocellulose membrane (NC) and filter paper of the same size as the gel strip were cut in advance and immersed in an electrotransport buffer. The gel was removed, after equilibration in transfer buffer, the membrane, gel and filter were sequentially laid, i.e., filter paper-NC membrane-gel-filter paper, and gently rolled through the sandwich with a clean glass rod to eliminate air bubbles between the layers. And (3) placing the sealed materials into an electrotransfer tank, adding electrotransfer liquid, and then connecting a cooling device, and transferring the 200mA constant current for 1h. Taking off NC membrane after transfer, washing membrane with TBST for 5min×3 times; placing NC membrane into 5% skimmed milk, sealing overnight at 4deg.C, discarding sealing solution, and washing membrane with TBST for 5min×3 times; adding primary antibody, namely diluting (1:100) African swine fever antibody positive pig serum with TBST, shaking smoothly, standing at room temperature for 1h, discarding the primary antibody, and washing the membrane with TBST for 5min multiplied by 4 times; adding horseradish peroxidase-labeled rabbit anti-pig enzyme-labeled secondary antibody, diluting with TBST at ratio of 1:50000, shaking smoothly at room temperature for 1 hr, discarding the secondary antibody, and washing the membrane with TBST for 5min×3 times. And placing the NC film acted by the secondary antibody into a color development liquid for color development, stopping the reaction after a specific reaction band appears, and taking a picture for storage.
The Western-blot analysis result shows that the African swine fever virus nonstructural protein dominant antigen epitope fusion protein can be identified by African swine fever virus positive serum, and has good immunoreactivity with the African swine fever virus positive serum as shown in figure 3.
Example 2 application of African swine fever virus nonstructural protein dominant antigen epitope fusion protein ELISA antibody detection kit in serological detection
An indirect ELISA method for detecting the African swine fever virus nonstructural protein antibody is established by utilizing the dominant antigen epitope fusion protein of the African swine fever virus nonstructural protein A238L prepared in the embodiment 1:
diluting the dominant epitope fusion protein of the African swine fever virus nonstructural protein A238L to 2.0 mug/mL by using a coating solution (0.05 mol/L carbonate buffer pH 9.6), adding the coating solution into an ELISA plate at the amount of 100 mug/hole, and coating at 4 ℃ overnight; PBST 300. Mu.L was added to each well, washed 2 times, and after drying, blocked with blocking solution (0.01 g/mL BSA) at 100. Mu.L/well for 2h at 37 ℃; adding 300 mu L of PBST into each hole, washing for 2 times, beating, adding 100 mu L of 1:100 diluted pig serum to be detected into each hole, simultaneously setting two holes (100 mu L/hole) of African swine fever virus antibody positive control sample and two holes (100 mu L/hole) of negative control sample, and discarding liquid in the holes after incubation at 37 ℃ for 30 min; adding 300 mu L of PBST into each well, washing for 5 times, beating, adding 100 mu L of HRP-labeled rabbit anti-pig IgG diluted with 1:50000 into each well, and discarding liquid in the well after 30min of action at 37 ℃; adding 300 mu L of PBST into each hole, washing for 5 times, beating to dry, adding 100 mu L of TMB single-component color development liquid into each hole, and developing for 10min in a dark place; finally, 100 mu L of 2mol/L H are added to each well 2 SO 4 Terminating the color development, and measuring the OD by using an enzyme-labeled instrument 450nm Values.
The result judgment standard of the kit detection method is as follows: when negative control OD 450nm Average value (N) less than 0.25, positive control OD 450nm When the average value (P) is greater than 0.65, the OD of the sample is 450nm Value (S)/negative control OD 450nm When the average value (N) is more than or equal to 2.1, the sample OD is positive 450nm Value (S)/negative control OD 450nm When the average value (N) is less than 2.1, the test is negative.
(1) The specificity of the detection method of the kit is measured
The results of the detection of the pig blue ear virus antibody, the pig circovirus antibody, the classical swine fever virus antibody, the pig foot-and-mouth disease virus antibody, the pig pseudorabies virus antibody, the pig encephalitis virus antibody, the pig parvovirus antibody, the haemophilus parasuis antibody and the pig type 2 streptococcus antibody positive pig serum by the detection method are shown in the table 1.
TABLE 1 results of the kit on the detection of common swine disease positive serum
Note that: positive control OD was detected at this time 450nm Average value of 1.197, negative control OD 450nm The average value of (2) is 0.214, the S/N is positive when the S/N is more than or equal to 2.1, and the S/N is less than 2.1, and the result is negative, "-": the result was negative.
The results in the table 1 show that the positive pig serum detection results of the porcine reproductive and respiratory syndrome virus antibody, the porcine circovirus antibody, the classical swine fever virus antibody, the porcine foot and mouth disease virus antibody, the porcine pseudorabies virus antibody, the porcine encephalitis virus antibody, the porcine parvovirus antibody, the haemophilus parasuis antibody and the porcine type 2 streptococcus antibody are all negative, and the kit has good specificity.
(2) Determination of sensitivity of the detection method
6 parts of African swine fever antibody positive swine serum are diluted according to the ratios of 1:50, 1:100, 1:200, 1:400, 1:800 and 1:1 600, and are respectively detected by the established indirect ELISA method for detecting the African swine fever virus nonstructural protein antibody and an African swine fever antibody ELISA detection kit produced by Spain INGENASA, wherein the detection results are shown in tables 2 and 3.
TABLE 2 results of the kit of this example on positive serum tests at different dilution ratios
Note that: positive control OD was detected at this time 450nm Average value of 1.298, negative control OD 450nm The average value of (2) is 0.227, positive when S/N is more than or equal to 2.1, negative when S/N is less than 2.1, "+": results were positive, "-": the result was negative.
TABLE 3 results of testing positive serum with Spain INGENASA kit for different dilution ratios
Note that: positive control OD was detected at this time 450nm Average value of 0.134, negative control OD 450nm The average value of (a) is 1.817, the sample blocking rate (%) = (negative control OD value-sample OD value)/(negative control OD value-positive control OD value) ×100%, the sample blocking rate (%) is positive when not less than 50%, the sample blocking rate (%) is negative when not more than 40%, and "+": results were positive, "-": the result was negative.
The results in Table 2 and Table 3 show that the serum titer of the kit disclosed by the invention is 1:400-1:800, the highest serum titer detected by the kit produced by the company Spain INGENASA is 1:50, and compared with the antibody titer of the kit produced by the company Spain INGENASA, the sensitivity of the kit disclosed by the invention for detecting the positive serum of African swine fever virus is obviously improved, the epidemic dynamics of the African swine fever virus in a pig farm can be accurately monitored, and important help is provided for early diagnosis and detection of the African swine fever virus.
(3) The kit is used for repeatedly measuring
3.1 In-batch reproducibility assay of the kit
30 parts of known background pig serum are detected by using the same batch of kit, wherein 15 parts of African swine fever antibody positive serum and 15 parts of African swine fever antibody negative serum are used. 3 replicates were performed for each of the 30 serum samples.
TABLE 4 results of 3 replicates of porcine serum samples tested with the same batch of kit
The results in Table 4 show that the intra-batch variation coefficient is between 0.49% and 6.96%, indicating that the intra-batch reproducibility of the kit is good.
3.2 Lot-to-lot repeatability test
The known background pig serum was tested for 30 parts using different batches of kit, of which 15 parts were african swine fever antibody positive serum and 15 parts were african swine fever antibody negative serum. The detection of 3 batch detection methods was performed simultaneously for each serum in 30 pig serum samples.
TABLE 5 results of testing pig serum with different lots of kit
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The results in Table 5 show that the inter-batch variation coefficient is between 0.37% and 8.84%, indicating that the inter-batch reproducibility of the kit is good.
(4) Comparison test with clinical application of the same kit
The above kit was compared with the kit manufactured by the company Ingenasa, spain, to detect 100 clinical pig serum.
The result shows that 100 parts of serum are detected by using the ELISA antibody detection kit for the african swine fever virus nonstructural protein dominant antigen epitope fusion protein, and the positive rate is 48.00% (48/100) and the negative rate is 52.00% (52/100); the positive rate was 41.00% (41/100) and the negative rate was 59.00% (59/100) as measured using the kit manufactured by the company Ingenasa, spain; the positive coincidence rate of the two is 85.42% (41/48), the negative coincidence rate is 88.14% (52/59), and the total coincidence rate is 93.00% (93/100).
EXAMPLE 3 preparation of high titer African swine fever Virus nonstructural protein antibodies
Three healthy clean New Zealand white rabbits, female, weighing 1.5-2 kg, are purchased from the medical laboratory animal center in Guangdong province, and two rabbits are selected to express the dominant antibody of the purified African swine fever virus nonstructural protein A238L by immunizationThe original epitope fusion protein and another rabbit were used as negative control. Immunization is carried out by adopting a back subcutaneous multipoint injection mode, the antigen epitope fusion protein of the African swine fever virus which expresses and purifies is mixed and emulsified with the equivalent Freund's complete adjuvant for the first immunization, each rabbit of an immunization group is inoculated with 1mL, the inoculated protein content is 180 mug/piece, and the mixture of the PBS and the Freund's complete adjuvant for the immunization of a negative control group is 1 mL/piece. In the subsequent immunization, the corresponding antigen is mixed and emulsified with Freund's incomplete adjuvant, and the mixture is treated in the same immunization dose and method, 15 days are separated every immunization, and the blood is collected on the 10 th day after the third immunization to separate serum for measuring the antibody titer. ELISA plates (100. Mu.L/well) were coated with expressed purified African swine fever virus epitope fusion protein (2.0. Mu.g/mL), washed 3 times with PBST, blocked at 100. Mu.L/well with 0.01g/mL BSA, after 2h at 37℃three rabbit sera were diluted sequentially with PBS at 1:1000 in a doubling ratio of 100. Mu.L/well, 30min at 37℃and 3 washes with PBST; HRP-labeled goat anti-rabbit enzyme-labeled secondary antibody 1:50000 was diluted, 100. Mu.L/well, allowed to act at 37℃for 30min, and washed 3 times with PBST; TMP monocomponent substrate was added at 100. Mu.L/well, developed at 37℃for 10min, and the reaction was stopped with 2M sulfuric acid stop solution at 100. Mu.L/well. Reading OD with an enzyme-labeled detector 450nm Values.
TABLE 6 determination of antibody titers of African swine fever virus epitope fusion proteins
Table 6 shows that when the serum of the experimental rabbits was diluted to 1:128,000, the two immunized rabbits corresponded to OD 450nm Value still negative control rabbit OD 450nm 2.1 times the value, the rabbit serum antibody titer of the present immunization epitope is 1:128 000.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The African swine fever virus nonstructural protein dominant antigen epitope fusion protein is characterized in that the amino acid sequence of the African swine fever virus nonstructural protein dominant antigen epitope fusion protein is shown as SEQ ID No. 1.
2. A nucleic acid encoding the african swine fever virus nonstructural protein dominant epitope fusion protein of claim 1.
3. The nucleic acid of claim 2, wherein the nucleic acid sequence is set forth in SEQ ID No. 2.
4. An expression vector comprising the nucleic acid of claim 2 or 3.
5. A host bacterium comprising the nucleic acid of claim 2 or 3 or the expression vector of claim 4.
6. An ELISA antibody detection kit for an African swine fever virus nonstructural protein dominant antigen epitope fusion protein, which is characterized by comprising an ELISA plate coated with the African swine fever virus nonstructural protein dominant antigen epitope fusion protein of claim 1.
7. The kit of claim 6, further comprising a coating solution, a washing solution, and a blocking solution.
8. The method for preparing the african swine fever virus nonstructural protein dominant antigen epitope fusion protein according to claim 1, which is characterized by comprising the following steps:
the expression vector of claim 4 is constructed, then the expression vector is transformed into host bacteria, after the host cells containing the African swine fever virus non-structural protein dominant antigen epitope fusion protein are induced to express, ultrasonic crushing, inclusion body dissolution and purification are carried out, and the African swine fever virus non-structural protein dominant antigen epitope fusion protein is obtained by collection.
9. A method for preparing an anti-african swine fever virus nonstructural protein antibody, which is characterized by comprising the following steps:
immunizing an animal with the dominant antigen epitope fusion protein as an antigen, and generating an african swine fever virus nonstructural protein antibody in the animal.
10. Use of the fusion protein of claim 1, the nucleic acid of claim 2, the expression vector of claim 4 or the host bacterium of claim 5 for the preparation of a product for detecting african swine fever virus.
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| CN103278627B (en) * | 2013-05-22 | 2015-09-02 | 扬州大学 | A kind of multi-antigen ELISA kit detecting African swine fever virus antibody |
-
2022
- 2022-08-31 CN CN202211058972.7A patent/CN116023506B/en active Active
- 2022-10-13 NL NL2033301A patent/NL2033301B1/en active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020102370A1 (en) * | 2018-11-15 | 2020-05-22 | Kansas State University Research Foundation | Immunogenic compositions for african swine fever virus |
| CN113543801A (en) * | 2019-03-27 | 2021-10-22 | 勃林格殷格翰动物保健有限公司 | Immunogenic composition and vaccine containing African swine fever virus peptide and protein and application thereof |
| CN114807178A (en) * | 2022-04-08 | 2022-07-29 | 广东省农业科学院动物卫生研究所 | C-terminal multi-epitope recombinant antigen of African swine fever virus P72 protein and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Analysis of the African Swine Fever Virus Immunomodulatory Proteins;Nefedeva M. V等;《Molecular Genetics, Microbiology and Virology》;20190115;第34卷(第1期);第42-49页 * |
| Mechanisms of African swine fever virus pathogenesis and immune evasion inferred from gene expression changes in infected swine macrophages;Zhu James J等;《PloS one》;20190414;第14卷(第11期);第1-22页 * |
| 非洲猪瘟病毒及其疫苗研究进展;黄霞等;《生物学杂志》;20211217;第38卷(第06期);第99-103页 * |
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| NL2033301B1 (en) | 2024-03-12 |
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