CN117164677A - Antigen peptide of porcine reproductive and respiratory syndrome virus NSP4 and polyclonal antibody thereof - Google Patents
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Abstract
The invention relates to the technical field of biology, and particularly discloses a polypeptide fragment of porcine reproductive and respiratory syndrome virus NSP4, a polyclonal antibody prepared by using the polypeptide fragment and application thereof. The amino acid sequences of the polypeptide fragments disclosed by the invention are shown as SEQ ID N0.1 and SEQ ID N0.2. The invention also discloses a preparation method of the polypeptide fragment and a polyclonal antibody prepared by using the polypeptide fragment. The polyclonal antibody prepared by the invention has higher sensitivity and specificity, can specifically identify NSP4 protein, can be used for detecting NSP4 protein, identifying protein functions, cell positioning and the like, provides important experimental materials for researching the biological functions of NSP4 protein, and lays a biological foundation for detecting porcine reproductive and respiratory syndrome virus.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antigenic peptide of porcine reproductive and respiratory syndrome virus NSP4 and a polyclonal antibody thereof.
Background
Since the end of the 80 s of the 20 th century, porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) has been found to be one of the most serious swine diseases worldwide, which is a virulent infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). PRRSV is a enveloped positive-strand RNA virus belonging to the arterivirus family that includes both PRRSV-1 (europe) and PRRSV-2 (north america) genotypes. PRRSV infects pigs to cause immunosuppression, and pregnant sows have abortion, mummy embryo and other reproductive disorders and diseases characterized by the piglet respiratory system.
The whole genome of PRRSV has 15kb, and at its 5' end there are two continuous open reading frames, ORF1a and ORF1b, respectively, encoding polyproteins pp1a and pp1ab, respectively, NSP4 is a non-structural protein of PRRSV, is a 3C-like serine protease, has the function of cutting virus macromolecular polyproteins into small molecular proteins, and is also necessary for replication of PRRSV. The NSP4 protein comprises three domains, the activation site of which is located between the domains I and II, and detection of the NSP4 protein can reflect the infection status of PRRSV, so that the preparation of antibodies capable of specifically detecting NSP4 is particularly important. Accordingly, the research on NSP4 protein has been increasing in recent years.
Chinese patent application CN116004551A discloses construction and application of a live vaccine for porcine reproductive and respiratory syndrome virus for regulating CIITA molecules, wherein the mutant porcine reproductive and respiratory syndrome virus is used for mutating a key amino acid site 199 of Nsp4 for regulating CIITA into aspartic acid. Nsp4 can degrade CIITA through ubiquitin proteasome pathway while cutting CIITA,199 sites are key sites for inhibiting CIITA, and V199D mutant virus can reduce inhibition of CIITA after vaccine strain infects bone marrow-derived dendritic cells to a certain extent, so that MHC-II molecule mediated antigen presenting pathway is restored.
Chinese patent No. 113943376B discloses a fusion gene, a coding protein thereof and application thereof in resisting African swine fever, wherein the fusion gene comprises at least one of an African swine fever virus nucleocapsid outer membrane protein CD2V gene and a virus envelope protein P17 gene and a surface protein HBHA gene of mycobacterium tuberculosis and a virulence gene NSP4 gene of rotavirus. Meanwhile, the invention also provides recombinant lactobacillus plantarum capable of expressing the protective antigen CD2V-P17-HBHA-NSP4, the recombinant lactobacillus plantarum has better growth performance compared with an original strain, has slower attenuation speed of live bacteria after a stabilization period, has good immune effect, can promote the generation of high-level CD8+ T cells in serum after virus attack, can obviously reduce the copy number of virus particles in serum, mouth, anus and excrement, effectively lightens pathological injuries of various organs in experiments, has good blocking effect on African swine fever virus, and can be used for preventing and treating African swine fever.
None of the above technical documents relates to the preparation of antigenic peptides and polyclonal antibodies from PRRSV NSP4 protein, and the present invention has been made based on this.
Disclosure of Invention
The invention aims to provide a polyclonal antibody of a porcine reproductive and respiratory syndrome virus NSP4 protein and application thereof in detecting the porcine reproductive and respiratory syndrome virus. According to the invention, through carrying out antigen peptide prediction on PRRSV NSP4 protein, two sections of antigen peptides respectively positioned in a structural domain I and a structural domain III are obtained, the applicant respectively synthesizes the antigen peptides by using an Fmoc solid phase method, and uses the obtained antigen peptides to immunize New Zealand big ear rabbits so as to obtain NSP4 polyclonal antibodies, and the antibodies can be effectively used for detecting PRRSV NSP4 proteins of different strains, thereby providing a powerful tool for carrying out research on PRRSV NSP4 and prevention, control and diagnosis of PRRSV in the future.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention claims an antigenic peptide of a non-structural protein 4 of a porcine reproductive and respiratory syndrome virus, wherein the antigenic peptide is positioned in a structural domain I or a structural domain III of NSP4 protein, and the amino acid sequence of the antigenic peptide is shown as SEQ ID NO. l or SEQ ID NO. 2.
The invention also claims a polyclonal antibody of antigen peptide of the porcine reproductive and respiratory syndrome virus NSP4, which is prepared by taking a composite protein formed by crosslinking the antigen peptide of the porcine reproductive and respiratory syndrome virus NSP4 after C-terminal modification with KLH carrier protein as an immunogen and immunizing an animal, wherein the animal is preferably New Zealand white rabbit.
The invention also claims a preparation method of the anti-porcine reproductive and respiratory syndrome virus NSP4 polyclonal antibody, which comprises the following steps:
a) Preparation of synthetic peptide antigen: artificially synthesizing amino acid sequences shown in SEQ ID No. l and SEQ ID No.2, and respectively performing coupling purification by using KLH carrier protein to obtain synthetic peptide antigens;
b) Immunization of New Zealand white rabbits: diluting antigen peptide by using normal saline, mixing and emulsifying the antigen peptide with Freund's adjuvant according to the ratio of 1:1, and immunizing New Zealand white rabbits to obtain serum containing polyclonal antibodies;
c) Purification of polyclonal antibodies: and purifying serum by adopting Protein G affinity chromatography to obtain the polyclonal antibody.
Preferably, in the preparation method of the anti-porcine reproductive and respiratory syndrome virus NSP4 polyclonal antibody, the immunization mode in the step b) is subcutaneous multipoint injection, and the immunization amount of the antigen peptide is 500 mug each time.
Preferably, in the method for preparing the anti-porcine reproductive and respiratory syndrome virus NSP4 polyclonal antibody, the step b) is performed with booster immunization every 14 days, and the serum containing the polyclonal antibody is obtained by intravenous or cardiac blood sampling at day 7 after six times of immunization.
Preferably, the anti-pig breeding and respiration combinationIn the preparation method of the virus NSP4 polyclonal antibody, the step b) comprises the detection of the antibody titer, and the titer and the sensitivity of the antibody are detected by an indirect ELISA method, wherein the titer of the polyclonal antibody is as high as 1:10 5 . The anti-porcine reproductive and respiratory syndrome virus NSP4 polyclonal antibody was stored in PBS buffer containing 20% glycerol.
The application of the polyclonal antibody against the porcine reproductive and respiratory syndrome virus NSP4 is used for Western Blot and indirect Immunofluorescence (IFA) detection of NSP4, and detection of NSP4 protein expression, and as a preferred mode, the polyclonal antibody is used for cell transient expression or expression detection of NSP4 protein infected by different strains of virus; alternatively, the polyclonal antibody is used for cellular localization of NSP 4.
Compared with the prior art, the invention provides two synthetic peptide fragments of porcine reproductive and respiratory syndrome virus NSP4, polyclonal antibodies, a preparation method and application, and has the following beneficial effects:
the invention has the beneficial effects that: the NSP4 polyclonal antibody meeting the requirement of subsequent experiments can be used for expression and cell location detection of NSP4 protein, and detection of PRRSV infection samples of different strains, and provides an effective tool for researching the functions of PRRSV NSP 4.
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 needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of transiently expressed Flag-NSP4 expressed proteins;
FIG. 2 is a graph showing Western Blot and IFA results of infection of Marc-145 cells at different time points at the same titer as HP-PRRSV;
FIG. 3 is a graph showing Western Blot and IFA results of infection of Marc-145 cells with different strains of PRRSV;
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The test methods used in the following examples are conventional methods unless otherwise specified: the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 preparation of polyclonal antibody against porcine reproductive and respiratory syndrome Virus NSP4
Polypeptide fragments having the amino acid sequences shown as SEQ ID No. l and SEQ ID No.2 of PNWQGVAPKAQFCED (selected antigenic polypeptide located at Dormin-1 of NSP 4) and VKPIKLSELSEFFAGP (selected antigenic polypeptide located at Dormin-3 of NSP 4), respectively, were selected as antigenic peptides of the polyclonal antibodies. Coupling on KLH carrier protein, purifying, diluting antigen peptide with normal saline, mixing with Freund's adjuvant according to 1:1 ratio, emulsifying, starting to immunize New Zealand white rabbit, taking vein blood as negative control before formal immunization, adopting subcutaneous injection or intramuscular injection, total amount of antigen is 500ug, boosting antigen peptide and Freund's incomplete adjuvant once every 14 days, taking vein or heart blood after six times immunization to obtain multiple antiserum. Serum was purified by Protein G affinity chromatography to give polyclonal antibodies, which were stored in 20% glycerol in PBS buffer.
Example 2 determination of antibody titers by indirect ELISA
Coating ELISA plates with the antigen peptide and KLH coupling protein, and coating at 4 ℃ overnight; the next day the plates were discarded, the plates were dried on absorbent paper, blocked with 2% BSA at room temperature (25 ℃) for 2h, two anti-NSP 4 polyclonal antibodies were diluted at different concentrations, 1:3125, 1:6250, 1:12500, 1:25000, 1:50000, 1:100000, negative control wells were used as controls, and non-immunized rabbit serum was taken for 1h at 37 ℃; after washing the plate, adding horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and incubating at 37 ℃ for 1h. After washing the plate, addTMB was subjected to a color reaction, and after termination of the reaction, the absorbance at a wavelength of 450nm was measured. The potency assay results are shown in tables 1 and 2: the titers of both polyclonal antibodies were as high as 1:10 5 The titer can ensure that the polyclonal antibody can be used for detecting NSP4 and ensure the quality of the antibody.
TABLE 1 ELISA titers detection of NSP4 Domain 1 synthetic peptide antibodies
Note that OD 450 >0.3, judging the antibody to be positive; OD (optical density) 450 <0.3, negative
TABLE 2 ELISA titers detection of NSP4 Domain 3 synthetic peptide antibodies
Note that OD 450 >0.3, judging the antibody to be positive; OD (optical density) 450 <0.3, negative
EXAMPLE 3 specificity analysis of polyclonal antibodies
To verify whether the resulting NSP4 protein synthetic peptide polyclonal antibody could be used in the subsequent Western Blot experiments, the resulting NSP4 protein synthetic peptide polyclonal antibody was used to detect the over-expression of Flag14-NSP4 plasmid in HEK 293T cells, by using murine anti-Flag antibody and two rabbit anti-NSP 4 polyclonal antibodies as primary antibodies, FIG. 1 is a graph of the results of the protein expressed by recombinant plasmid Flag14-NSP4, as shown in FIG. 1, both polyclonal antibodies NSP4D 1 and NSP4D3 could specifically recognize the transiently post-transfected NSP4 protein, and fewer non-specific bands of NSP4D3 could be considered for the subsequent validation experiments as compared to NSP4D 1. Thus, this result demonstrates that two rabbit anti-NSP 4 polyclonal antibodies can be prepared for NSP4 detection by transient cell expression, and that polyclonal antibody NSP4D3 has better specificity than polyclonal antibody NSP4D 1.
EXAMPLE 4 verification of infection of Marc-145 cells at different time points with HP-PRRSV at the same titer
Spreading Marc-145 cells with good state on a 60mm cell culture dish, incubating 0.5MOI HP-PRRSV (SH 01) and the cells for 2h, changing into 2% DMEM culture medium, continuously culturing, collecting cell samples after virus infection for 12h, 24h and 48h, and setting an uninfected group as a control group. Protein expression was detected by using the rabbit anti-NSP 4 polyclonal antibody NSP4D3, the rabbit anti-PRRSV N protein antibody and the murine anti-beta-actin antibody as primary antibodies, the detection results are shown in FIG. 2, and the rabbit anti-NSP 4 polyclonal antibody can specifically detect a band of about 20kDa after 12 hours of virus infection, the size of which is consistent with that predicted, and the protein content increases with the time of virus infection. In addition, the samples tested were rechecked with antibodies against PRRSV N protein, which was detected 12h after viral infection (fig. 2A). Further, the results of IFA detection showed that virus-infected cells could be specifically detected using the rabbit anti-NSP 4 polyclonal antibody NSP4D3, while only fluorescence-positive cells were present in the infected cells compared to the uninfected cell samples, and the number of these cells increased with prolonged virus infection time; meanwhile, the results of the rabbit anti-NSP 4 polyclonal antibody were identical to those of the anti-N protein (fig. 2B). Thus, these results demonstrate that the prepared rabbit anti-NSP 4 polyclonal antibodies can be used for NSP4 detection generated during viral infection.
EXAMPLE 5 verification of infection of Marc-145 cells with different strains of PRRSV
To verify whether the resulting NSP4 protein synthetic peptide polyclonal antibody NSP4D3 can detect NSP4 proteins of cells infected with different source strains, a large amount of NSP4 proteins were expressed in the cells in view of 24 hours after virus infection. Thus, highly pathogenic strain (SH 01), vaccine strain (JX 1R) and classical strain (CHIR) were selected to infect Marc-145 cells at a viral load of 0.5MOI, and samples were prepared and analyzed 24h after infection. The results of Western Blot showed that NSP4 antigen peptide polyclonal antibody NSP4D3 can specifically detect NSP4 protein expression in SH01, JX1R and CHIR virus infected samples (FIG. 3A). In addition, the results of IFA showed that the antigen peptide polyclonal antibody can effectively detect virus-infected cells, and that the staining results of NSP4D3 antibodies on SH01, JX1R and CHIR-infected cells can overlap with the N protein detection image (fig. 3B). Thus, these results further demonstrate that the prepared rabbit anti-NSP 4 polyclonal antibodies can be used for the detection of NSP4 expression in highly pathogenic strain (SH 01), vaccine strain (JX 1R) and classical strain (CHIR) infected samples.
Claims (10)
1. An antigenic peptide of porcine reproductive and respiratory syndrome virus NSP4 is positioned in a structural domain I or a structural domain III of NSP4 protein, and the amino acid sequence of the antigenic peptide is shown as SEQ ID NO. l or SEQ ID NO. 2.
2. The polyclonal antibody of the NSP4 polypeptide of the porcine reproductive and respiratory syndrome virus is characterized in that the polyclonal antibody is prepared by taking a composite protein formed by cross-linking the antigen peptide of the NSP4 of the porcine reproductive and respiratory syndrome virus after C-terminal modification with a KLH carrier protein as an immunogen and immunizing an animal, wherein the animal is preferably New Zealand white rabbit.
3. A method of preparing the anti-porcine reproductive and respiratory syndrome virus NSP4 polyclonal antibody according to claim 2, comprising the steps of:
a) Preparation of synthetic peptide antigen: artificially synthesizing an amino acid sequence shown as SEQ ID NO. l or SEQ ID NO.2, and respectively performing coupling purification by using KLH carrier protein to obtain a synthetic peptide antigen;
b) Immunization of New Zealand white rabbits: diluting antigen peptide by using normal saline, mixing and emulsifying the antigen peptide with Freund's adjuvant according to the ratio of 1:1, and immunizing New Zealand white rabbits to obtain serum containing polyclonal antibodies;
c) Purification of polyclonal antibodies: and purifying serum by adopting Protein G affinity chromatography to obtain the polyclonal antibody.
4. The method for preparing the polyclonal antibody against the porcine reproductive and respiratory syndrome virus NSP4 according to claim 3, further comprising a potency detection step.
5. The method for preparing the anti-porcine reproductive and respiratory syndrome virus NSP4 polyclonal antibody according to claim 3, wherein the titer of the anti-porcine reproductive and respiratory syndrome virus NSP4 polyclonal antibody is detected by an indirect ELISA method.
6. The method for preparing the polyclonal antibody against the NSP4 of the porcine reproductive and respiratory syndrome virus according to claim 3, wherein the immunization mode in the step b) is subcutaneous multipoint injection, and the immunization amount of the antigen peptide is 500 mug each.
7. The method for preparing polyclonal antibody against NSP4 of porcine reproductive and respiratory syndrome virus according to claim 3, wherein the step b) is performed once every 14 days, and serum containing polyclonal antibody is obtained by intravenous or cardiac blood sampling at day 7 after six times of immunization.
8. The method for preparing polyclonal antibody against NSP4 of porcine reproductive and respiratory syndrome virus according to claim 3, wherein the titer and sensitivity of the polyclonal antibody in step d) are detected by indirect ELISA, and the titer of the polyclonal antibody is as high as 1:10 5 The anti-porcine reproductive and respiratory syndrome virus NSP4 polyclonal antibody was stored in PBS buffer containing 20% glycerol.
9. Use of the polyclonal antibody against NSP4 of porcine reproductive and respiratory syndrome virus according to claim 2 for the detection of NSP4 protein expression of porcine reproductive and respiratory syndrome virus.
10. The use of polyclonal antibodies against NSP4 of porcine reproductive and respiratory syndrome virus according to claim 7, wherein said polyclonal antibodies are used for the detection of the expression of NSP4 proteins of different strains.
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