CN117903262A - Preparation, epitope identification and application of porcine reproductive and respiratory syndrome virus Nsp7 monoclonal antibody - Google Patents
Preparation, epitope identification and application of porcine reproductive and respiratory syndrome virus Nsp7 monoclonal antibody Download PDFInfo
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Abstract
The invention relates to preparation, epitope identification and application of a porcine reproductive and respiratory syndrome virus Nsp7 monoclonal antibody. Through constructing prokaryotic expression vector pCold-I-NSp7, expressing recombinant NSp7 by using colibacillus, immunizing Balb/c mouse after purification, taking spleen cells of the mouse after four times of immunization, fusing with myeloma SP2/0 cells to prepare hybridoma cells, and then subcloning by using an indirect ELISA method to obtain monoclonal antibody of anti-NSp 7, and identifying that the epitope corresponding to the antibody is 184KFWDKNSGD192.
Description
Technical Field
The invention relates to the field of biology, and particularly provides a preparation method, epitope identification and application of a monoclonal antibody aiming at PRRSV Nsp 7.
Background
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a single-stranded positive-stranded enveloped RNA virus belonging to the order of the nipaginales, genus arterivirus, family arteriviridae. PRRSV has stringent host specificity and tissue tropism, and primarily infects alveolar macrophages and dendritic cells, causing acute or persistent infections. Porcine alveolar macrophages are the primary target cells for PRRSV replication in vivo in respiratory tract infections. The PRRSV genome is approximately 15kb and includes a cap structure and untranslated region (UTR) at the 5 'end, and a UTR and poly (a) tail at the 3' end. Between the 5 'and 3' ends, PRRSV encodes at least 10 Open Reading Frames (ORFs), ORF1a, ORF1b, ORF2a, ORF2b, ORFs 3-7 and ORF5a, respectively. ORF1a and ORF1b represent three quarters of the viral genome, encoding the replicase multimeric proteins pp1a, pp1a-Nsp2N, pp a-Nsp2TF and pp1ab mainly by ribosomal frameshifting. These multimeric proteins are processed hydrolytically into at least 16 functional nonstructural proteins (Nsp), including Nsp1 alpha, nsp1 beta, nsp2TF, nsp2N and Nsp 2-6, nsp7 alpha, nsp7 beta and Nsp 8-12, responsible for replication and transcription of the viral genome.
The full length of the Nsp7 coding gene is 777bp, consists of 259 amino acids (aa), is highly conserved among different PRRSV strains, has no obvious sequence difference, and can be further cut into Nsp7 alpha and Nsp7 beta in vivo. The research shows that NSp7 has good immunogenicity and can be used for serological detection of PRRSV specific antibodies. After pigs are infected with PRRSV, NSP1, NSP2 and NSP7 are three non-structural proteins with highest immune activity. Nsp7 antibodies were produced earlier, at higher levels and for longer durations than N protein antibodies, indicating that Nsp7 plays a very important role in the humoral immune response induced by PRRSV infection. At present, the detection target of the commercial PRRSV antibody detection kit of Edison is N protein antibody, and the ELISA detection kit aiming at non-structural proteins is relatively deficient. The Nsp7 is only expressed in the virus replication process, so that the inactivated vaccine immunized pig does not produce Nsp7 antibodies, and the attenuated vaccine immunized pig and the wild virus infected pig can produce Nsp7 antibodies, so that ELISA detection aiming at the Nsp7 antibodies can also play a role in differential diagnosis. In conclusion, the preparation of the monoclonal antibody aiming at Nsp7 and the identification of the identified epitope thereof are helpful for the design and development of PRRSV antibody detection reagents and the development of novel genetic engineering vaccines, and have very important application prospects.
The invention successfully obtains a specific monoclonal antibody aiming at PRRSV Nsp7 by utilizing cell fusion and subcloning screening, and identifies the identified epitope thereof, thereby providing a valuable tool for the basic and application research of PRRSV in the future.
Disclosure of Invention
Aiming at the blank existing in the prior art, the invention provides a monoclonal antibody of PRRSV Nsp7 of pigs, and a preparation method and application thereof. The technical scheme of the method is as follows: the coding sequence of PRRSV NSp7 is cloned into pCold-I plasmid to construct prokaryotic expression vector, the protein is purified by means of eluting with imidazole with different concentrations, the purified NSp7 is used as antigen to immunize Balb/c mouse, spleen cells of the mouse are fused with SP2/0 cells of myeloma cells to prepare hybridoma cells, the purified NSp7 is used for coating plates, and positive hybridoma cells capable of specifically recognizing NSp7 are screened by indirect ELISA.
Specifically, the invention aims to provide an epitope of a porcine reproductive and respiratory syndrome virus nonstructural protein 7 (PRRSV Nsp 7), wherein the epitope is 184-192 peptide of PRRSV Nsp7, and the amino acid sequence of the epitope is (184KFWDKNSGD192).
The invention also aims to provide the application of the epitope of the porcine reproductive and respiratory syndrome virus nonstructural protein 7 (PRRSV NSp 7) in the preparation of porcine reproductive and respiratory syndrome virus epitope vaccine antigen and diagnostic reagent antigen.
The invention also aims to provide a method for determining and preparing the epitope of the porcine reproductive and respiratory syndrome virus nonstructural protein 7 (PRRSV NSp 7).
Advantageous effects
The invention obtains 1 monoclonal antibody capable of specifically recognizing PRRSV Nsp7, which can be used for IFA and Western blotting detection, and can well recognize Nsp7, the subtype heavy chain of the monoclonal antibody is IgG2a, and the light chain is Kappa. By constructing truncated Nsp7 and synthesizing a series of Nsp7 short peptides, the 184-192 peptide segment of the monoclonal antibody capable of specifically recognizing the Nsp7 is finally determined, and the amino acid sequence of the monoclonal antibody is 184KFWDKNSGD192, as shown in SEQ ID NO. 1.
Drawings
FIG. 1 is a PCR amplification electrophoresis of a PRRSV Nsp7 coding gene fragment in example 1 of the present invention. Wherein, the M lane is DNA standard DL2000, the 1 lane is PRRSV Nsp7 coding gene fragment, which accords with the expected 777bp, which indicates that the fragment with correct size is successfully obtained.
FIG. 2 is a Western blotting identification chart of the protein purified by Coomassie brilliant blue staining induced by Nsp7 in example 2.
Wherein, lane M is a protein molecular mass standard, lane1 is a pCold-I empty vector control, lane 2 is a whole bacterium before pCold-I-NSp7 induction, lane 3 is a whole bacterium after pCold-I-NSp7 induction, lane 4 is a supernatant after pCold-I-NSp7 induction, and lane 5 is a precipitate after pCold-I-NSp7 induction, which indicates that recombinant NSp7 is mainly expressed in the supernatant after induction and can be used for purifying antigen proteins. Lane 6 is coomassie brilliant blue staining identification of the recombinant Nsp7 antigen protein after purification, lane 7 is a blank control, lane 8 is Western blotting identification of the recombinant Nsp7 antigen protein after purification by using His tag antibody, which shows that the protein has a good purification effect, the band size accords with the expected value, about 35kDa, and can specifically react with the His tag, thus showing that the purified protein is correct and can be used for later immunization.
FIG. 3 is an IFA map of PRRSV infected MARC-145 cells detected with the 3A3 monoclonal antibody of example 5, demonstrating that the 3A3 antibody does not fluoresce when reacted with blank cells, but can specifically recognize PRRSV infected cells, producing green fluorescence.
FIG. 4 is a Western blotting identification chart of example 5 using 3A3 monoclonal antibody, PRRSV N protein antibody and beta-actin internal reference antibody, respectively, for different times after PRRSV infection of MARC-145 cells, showing that 3A3 antibody can recognize PRRSV NSp7 and produce specific band, and that the NSp7 expression level is gradually increased at 12h, 24h and 36h after PRRSV infection, consistent with the expression trend of PRRSV N protein.
FIG. 5 is A3A 3 monoclonal antibody subtype assay of example 5, illustrating the heavy chain of this antibody being IgG2a and the light chain being Kappa.
FIG. 6 is a Western blotting diagram of the epitope of the preliminary fragment screening 3A3 monoclonal antibody of example 6, illustrating that the antibodies recognize truncated Nsp7 fragments each comprising the 184KFWDKNSGD192 peptide.
FIG. 7 is an ELISA identification of the smallest epitope in example 6, demonstrating that the 3A3 monoclonal antibody recognizes the smallest epitope as 184KFWDKNSGD192 peptide.
Detailed Description
The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
EXAMPLE 1 construction of recombinant prokaryotic expression vector pCold-I-NSp7
The inventors first constructed pCold-I-NSp7 prokaryotic expression plasmid and designed the primers as follows:
Nsp7-F：5′－GGGGTACCTCGCTGACTGGTGCCCTCGC－3′(SEQ ID NO.2)
Nsp7-R：5′－CGGGATCCTTATTCCCACTGAGCTCTTTTAT－3′(SEQ ID NO.3)
The primers are all pCold-I-NSp7 specific sequences through BLAST analysis, and are synthesized by Shanghai qing biological science and technology Co. The template is the gene sequence of PRRSV vHuN strain (GenBank: EF 635006.1) coding NSp7 in reference NCBI, and is connected into pCold-I vector, and is completely identical with the gene sequence of PRRSV NSp7 by sequencing and comparison, and is named pCold-I-NSp7. The specific PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s; denaturation 95 ℃,10s, annealing 65 ℃,30s, extension 72 ℃,1min,30 cycles; 72℃for 10min. After electrophoresis, the gel was observed under ultraviolet light and photographed. The synthesized plasmid was identified by PCR using the designed primers, and after electrophoresis, the specific band of about 777bp was amplified by ultraviolet observation (FIG. 1), and the result was consistent with the expectation.
EXAMPLE 2 inducible expression and purification of recombinant prokaryotic expression vector pCold-I-NSp7
BL21 competent cells were transformed with pCold-I-NSp7 and pCold-I empty vector, uniformly spread on LB solid agar medium with ampicillin resistance, and cultured upside down in a constant temperature incubator at 37 ℃. Individual colonies were randomly picked and inoculated into LB liquid medium with ampicillin resistance and shaken overnight in a shaking table at a constant temperature of 37 ℃. The next day, 1mL of each bacterial liquid is added into two new LB liquid culture media with ampicillin resistance, and shaking culture is carried out at a constant temperature of 37 ℃. When the bacterial liquid OD 600nm is about 0.6-0.8, IPTG is added to one of the tubes as an induction group. The other group was not treated and was a control group. And (5) carrying out low-temperature induction at 16 ℃ for 16 hours, and collecting samples. The induced group and the control group were collected by centrifugation with 1mL of each bacterial liquid, and the bacterial cells were treated and subjected to SDS-PAGE electrophoresis to analyze the induced expression of the plasmid pCold-I-NSp7 (FIG. 2). After successful identification, the bacterial liquid remained in the induction group is centrifuged at a high speed, then the bacterial liquid is collected and subjected to ultrasound, SDS-PAGE electrophoresis is carried out, and whether the protein is distributed in the supernatant or inclusion body after the induction of the recombinant prokaryotic plasmid pCold-I-NSp7 is analyzed. As shown in FIG. 2, the recombinant proteins are distributed in the supernatant. Nsp7 was purified according to the affinity chromatography principle using a nickel column from QIAGEN. The method comprises the following steps: collecting a large amount of Nsp7 bacterial liquid with His tag expression, washing with sterile PBS for 2 times, crushing by using an ultrasonic crusher, centrifuging at high speed, and collecting the supernatant. The supernatant was combined with a pretreated nickel column overnight at 4 ℃. The nickel column was washed with washing solutions containing 10mM, 30mM, 50mM, 100mM, 200mM and 400mM imidazole, and the eluate was collected and subjected to SDS-PAGE electrophoresis, and the purification result was observed. After SDS-PAGE electrophoresis of the purified proteins, the purified proteins were transferred to NC membrane for Western blotting experiments, and reaction specificity of the purified proteins was identified by His tag antibodies (FIG. 2).
EXAMPLE 3 animal immunization
Purified Nsp7 antigen protein was immunized with 4 female Balb/C mice of 5 weeks of age, and another set of 4 mice was used as negative control. The concentration of purified protein was determined prior to immunization. 100 mug of purified protein is taken to be emulsified with an equal volume of Freund's complete adjuvant, the mice are immunized by a back subcutaneous injection method, after two weeks, the mice are injected into the bodies of the mice by the same way after the Freund's incomplete adjuvant is emulsified with the protein, and the mice are immunized for a third time after four weeks. Three days after the third immunization, the tail of the mice is cut off, the serum antibody titer is measured by ELISA, and the two detection results reach the standard and then the mice are subjected to the boosting immunization.
EXAMPLE 4 preparation of PRRSV Nsp7 monoclonal antibody
(1) Culturing SP2/0 cells
SP2/0 cryopreserved cells were removed from liquid nitrogen 7 days prior to fusion and rapidly placed in a 37℃water bath with shaking for thawing. After complete thawing, the supernatant was discarded in a biosafety cabinet by centrifugation at 1000rpm for 5min, the pellet was blow-mixed with 1mL of 10% serum-containing DMEM medium, spread onto a cell culture dish, added with medium to 10mL, and incubated in a 5% CO 2 incubator at 37 ℃.
(2) Preparation of feeder cells
The day before fusion, serum is taken as a negative control from mice in a control group by adopting an eyeball blood sampling method, and the mice are dead and soaked in 75% alcohol for disinfection. The abdomen of the mouse is fixed on a sterilized foam board in a biosafety cabinet upwards, after the skin of the abdomen is clamped by forceps, the skin at the position of the fat layer below the abdomen of the mouse is sheared into a small hole, and the skin is torn from the small hole by the forceps and the scissors to expose the abdominal cavity. 5mL of DMEM medium was injected intraperitoneally. The peritoneal membrane was carefully flicked on both sides with forceps to suspend macrophages in the culture medium, and the medium was aspirated after several flicks. This procedure was repeated, and the aspirated culture broth was dispensed into 96-well plates at 100. Mu.L per well. The next day, the growth state of feeder cells was observed, and the feeder cells were fused when the growth state was good.
(3) Preparation of spleen cells
Serum was taken as a positive control from immunized mice by taking blood from the eyeballs, and the mice were sacrificed and sterilized by soaking in 75% alcohol. The mice were fixed, the abdominal skin of the mice was gently cut off with sterile scissors, and spleens were picked up under aseptic conditions and placed in petri dishes. The separated complete spleen was carefully pricked with a 5mL sterile syringe at one end of the spleen with a few small holes, and after the basal culture solution was sucked with a 5mL sterile syringe at the other end, the spleen was carefully pricked and washed with the spleen, and repeated a plurality of times until the spleen became off-white, and the spleen cells were completely washed out.
(4) Cell fusion
Mixing SP2/0 myeloma cell suspension and immune spleen cell suspension according to a ratio of 1:3, placing into a 50ml centrifuge tube, adding DMEM to 30ml, fully and uniformly mixing, centrifuging cells at 1000rpm for 10min, discarding the supernatant, tapping the bottom of the tube to disperse cell sediment into paste, placing the centrifuge tube filled with spleen cells and SP2/0 cells into a foam box filled with warm water at 37 ℃, rotating the centrifuge tube, slowly dripping 1ml of PEG2000 preheated in a 37 ℃ incubator, standing for 2min after the adding is completed. And then, the DMEM basal culture solution is slowly dripped to dilute the PEG to terminate fusion, and the mixture is centrifuged at 1000rpm for 10min. Cells were resuspended in HAT medium containing 20% serum pre-warmed at 37℃and added to 96-well cell culture plates plated with feeder cells at a volume of 100. Mu.L/well and incubated in a 5% CO 2 incubator at 37 ℃. After 10 days of fusion, 50. Mu.L of cell culture supernatant was carefully aspirated with a row gun and positive cell wells secreting antibodies were screened by indirect ELISA.
(5) Screening positive hybridoma cells
The supernatant of the fused cells is used as a primary antibody, the supernatant of the SP2/0 cells is used as a negative control, the serum of the mice before fusion is used as a positive control, and the goat anti-mouse IgG marked by HRP is used as a secondary antibody. The specific implementation method is as follows: ELISA plates were coated with purified Nsp7 antigen protein at 400 ng/well, 100. Mu.L per well, and 4℃overnight. 200 mu L of 5% skim milk is added into each hole and is blocked for 2 hours at 37 ℃, after the mixture is washed 3 times by PBST containing 5 permillage Tween-20, 100 mu L of cell supernatant is added into an ELISA plate coated with protein and is uniformly mixed, after the incubator acts for 1 hour at 37 ℃, the mixture is washed 3 times by PBST containing 5 permillage Tween-20 for 5 minutes each time. 100. Mu.L of secondary antibody diluted in a ratio of 1:10000 was added thereto, and after incubation at 37℃for 45min, the solution was washed 3 times with PBST containing 5% Tween-20. 100. Mu.L of TMB color development solution was added to each well, and incubated at room temperature for 15min in the dark. The reaction was stopped by adding 50. Mu.L of 2M H 2SO4 to each well and the OD 450nm was measured by a microplate reader. After the positive cell hole with the highest OD value is selected for amplification culture, 3 subclones are carried out, and each subclone is screened by the indirect ELISA method. And selecting a hybridoma cell strain 3A3 which stably secretes the Nsp7 antibody for carrying out expansion culture and preparing ascites.
(6) Preparation of ascites
Freund's incomplete adjuvant was intraperitoneally injected 3 days in advance into 8-week-old Balb/c female mice, each with 0.2mL. After one week, hybridoma cells well grown were blown up and transferred to a 15mL centrifuge tube at 1000rpm for 5min. After the cell pellet was resuspended in PBS, mice were intraperitoneally injected with 10 6 cells each. After 7 days, the mice were distended in the abdominal cavity, ascites were collected, centrifuged and the supernatant was aspirated and stored frozen at-80 ℃.
Example 5: specific detection and subtype identification of monoclonal antibodies
To verify the specificity of the monoclonal antibodies, PRRSV vHuN strain was used to infect MARC-145 cells and IFA analysis was performed. The results show that the 3A3 monoclonal antibody specifically recognizes PRRSV-infected cells and fluoresces green, whereas the control group does not (fig. 3). The PRRSV vHuN.sup.4 strain was used to infect MARC-145 cells, and the proteins were collected at 12h, 24h and 36h after infection and subjected to Western blotting analysis. The results showed that the 3A3 monoclonal antibody recognizes that the cells after virus infection produced specific protein bands, the bands were clear, and the expression level of Nsp7 increased with the infection time (FIG. 4). The identification of the antibody subtype was performed using Proteintech monoclonal antibody subtype identification kit, which showed that the heavy chain was IgG2a and the light chain was Kappa (fig. 5). Subsequently, the collected ascites was purified by using Protein G, and the purified 3A3 monoclonal antibody titer was measured as 1:512000 by an indirect ELISA method.
EXAMPLE 6 identification of epitope recognized by Nsp7 monoclonal antibody
The Nsp7 (1-259 aa) was first divided into two segments (Nsp 7α: 1-149 aa; nsp7β: 150-259 aa), and expression plasmids were constructed according to the primers (SEQ NO. 2-5) in Table 1, and the schematic fragment is shown in FIG. 6A. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 30s; denaturation 95 ℃,10s, annealing 65 ℃,30s, extension 72 ℃,30s,30 cycles; 72℃for 10min. Connecting the fragments to pCold-I vector, transforming BL21 competence, selecting positive clone, selecting single colony for verification, performing induction expression after verification is correct, culturing bacteria in a shaking table at 37 ℃ until OD value reaches 0.6-0.8, adding 1mM IPTG for induction expression, performing induction at 16 ℃ for 20h, and collecting bacterial liquid for ultrasound. The results indicated that the monoclonal 3A3 epitope is located within the Nsp7β region (150-259 aa) (FIG. 6B). The Nsp7 alpha fragment was further truncated and divided into four segments (Nsp 7 beta-1:150-189 aa; nsp7 beta-2:184-224 aa; nsp7 beta-3:219-259 aa), amplified as described above based on the primers in Table 1 (SEQ No.5-9 and SEQ No. 3), ligated into pCold-TF vector, BL21 competent was transformed, positive clones were selected, and expression characterization was induced. The results indicate that the linear epitope recognized by this monoclonal antibody 3A3 is located in the Nsp7 beta-2 region (184-224 aa) (FIG. 6C).
TABLE 1 primer sequences used in the patents of the present invention
To further accurately identify the epitope of the 3A3 monoclonal antibody, 8 different truncated polypeptides (SEQ NO.1 and SEQ NO. 10-16) were synthesized by Jier Biochemical (Shanghai) Inc., and the 8 polypeptides were coated with ELISA plates, respectively, for indirect ELISA identification. The results indicate that the 3A3 monoclonal antibody recognizes the minimum epitope of Nsp7 as a stretch of 184-192 amino acids and has the sequence 184KFWDKNSGD192 (FIG. 7).
Example 7 establishment of Nsp7 Indirect ELISA method and results of conditional optimization
(1) Determination of optimal coating concentration of antigen and optimal serum dilution
Purified Nsp7 antigen protein was diluted to different concentrations for coating, PRRSV positive and negative sera were diluted from 20-fold to 200-fold, respectively, and the results of the square titration showed (table 2) that the optimal coating amount of antigen was 4 μg/mL (400 ng/well), the dilution of serum was 1:50, and the OD 450nm values of positive and negative sera at this time differed the most (P/n= 12.735).
TABLE 2 determination of optimal coating concentration of antigen and optimal dilution of serum
(2) Optimal coating liquid, optimal sealing liquid and optimal sealing time result
The ELISA plates were coated with 4. Mu.g/mL (400 ng/well) of the different coating solutions and blocked with different blocking solutions for different times, which indicated (Table 3) that the P/N value was the maximum (P/N= 11.903) when the antigen was coated with the phosphate coating solution and blocked with 5% skim milk at 37℃for 2h, as the optimal condition.
TABLE 3 determination of optimal antigen coating fluid and blocking conditions
(3) Coating temperature time and enzyme-labeled secondary antibody dilution optimization result
Coating antigen with phosphate coating liquid at 37 ℃ for 1h, 37 ℃ for 2h, 37 ℃ for 4h and 4 ℃ for overnight respectively, wherein the result shows that the P/N value is maximum when the coating liquid is coated at 4 ℃ for overnight; the enzyme-labeled secondary antibodies were diluted 5×10 3、1×104、2×104、4×104 times, and then applied at 37℃for 1 hour, which showed that the P/N value was the largest (P/N= 16.266) when diluted 1×10 4 times, and the optimal secondary antibody dilution was determined to be 1×10 4.
TABLE 4 determination of optimal coating method for antigen and secondary antibody dilution
(4) Optimization results of optimal incubation time of serum and enzyme-labeled secondary antibody
Experiments were performed according to the above-defined conditions, and after the serum was incubated for four times of 20min, 30min, 45min, and 60min, the diluted second antibody was incubated for four times of 20min, 30min, 45min, and 60min, and the results showed (table 5) that the optimal serum incubation time was 60min and the optimal second antibody incubation time was 60min.
TABLE 5 determination of serum optimal reaction time and secondary antibody optimal reaction time
(5) Optimization of color development temperature and time
According to the experiment under the conditions, the color development conditions are respectively room temperature and 37 ℃, the color development time is 5min, 10min and 15min, ELISA measurement is carried out, and the result shows that (table 6) the color development is carried out at the room temperature for 15min, and the color development temperature time of the optimal substrate is reached, and the P/N value can reach 12.462.
TABLE 6 determination of optimal substrate color conditions
(6) Determination of the threshold of the Indirect ELISA method
The results were statistically analyzed (Table 7) by taking 27 PRRSV negative serum samples of known background stored in the laboratory and testing them under optimized indirect ELISA conditions, and the average value was calculated to be 0.160 and the standard deviation was calculated to be 0.045. When OD 450nm is more than or equal to X+3SD=0.295, the serum is judged positive; when OD 450nm is less than or equal to x+2sd=0.25, the serum is judged as negative, and the suspicious serum is between the two.
TABLE 7 ELISA test results for 27 pig negative serum
In conclusion, the invention provides an epitope aiming at PRRSV Nsp7, a monoclonal antibody preparation method and application. PRRSV NSP7 was obtained by His tag purification by cloning the PRRSV NSP7 coding sequence into a pCold-I vector to construct a prokaryotic expression vector capable of expressing PRRSV NSP7. Balb/c mice were immunized with the purified protein as an immunogen, and monoclonal antibodies against PRRSV Nsp7 were obtained by cell fusion and subcloning screening. The identification result shows that the heavy chain of the prepared antibody subtype is IgG2a, and the light chain is Kappa. IFA and Western blotting analysis results show that the obtained monoclonal antibody 3A3 can specifically identify PRRSV Nsp7 and can be used for detecting PRRSV Nsp7 under reducing conditions, and the identified epitope sequence is 184KFWDKNSGD192, so that the specificity of the antibody is further demonstrated.
It should be understood that while the invention has been described in detail with respect to the general description and specific embodiments thereof, it will be apparent to those skilled in the art that certain modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (6)
1. An epitope of a porcine reproductive and respiratory syndrome virus nonstructural protein 7 (PRRSV NSP 7), wherein the epitope is 184-192 peptide segment of PRRSV NSP7, and the amino acid sequence is (184KFWDKNSGD192).
2. The use of an epitope of porcine reproductive and respiratory syndrome virus nonstructural protein 7 (PRRSV Nsp 7) according to claim 1 in the preparation of porcine reproductive and respiratory syndrome virus epitope vaccine antigens and diagnostic reagent antigens.
3. An antibody preparation comprising an antibody specific for an epitope of PRRSV Nsp7 as defined in claim 1, said antibody being monoclonal; or a fragment of said antibody.
4. A method of preparing an antisera, the method comprising administering the epitope of claim 1 to an animal host to produce antibodies in the animal host and recovering the antisera comprising the antibodies produced in the animal host.
5. An antigen composition comprising at least one antigen, wherein the at least one antigen comprises an epitope of porcine PRRSV Nsp7, wherein the epitope of porcine PRRSV Nsp7 is located at the 184-192 peptide stretch, and the amino acid sequence of the epitope is shown in SEQ ID No.1 (184KFWDKNSGD192).
6. Use of an epitope of porcine PRRSV Nsp7 according to claim 1, an antibody preparation according to claim 3, an antigen composition according to claim 5 for the preparation of an ELISA antibody detection kit for diagnosis or detection of PRRSV.
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| CN102483411A (en) * | 2008-11-26 | 2012-05-30 | 南达科他州立大学 | Identification of porcine reproductive and respiratory syndrome virus |
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| CN102483411A (en) * | 2008-11-26 | 2012-05-30 | 南达科他州立大学 | Identification of porcine reproductive and respiratory syndrome virus |
| US20160116471A1 (en) * | 2013-05-15 | 2016-04-28 | Prionics Ag | Method for the detection and classification of prrsv-infections in swine herds and diagnostic antigen compositions for such methods |
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