CN117343149A - Preparation and application of PRRSV nonstructural protein Nsp2 synthetic peptide polyclonal antibody - Google Patents
Preparation and application of PRRSV nonstructural protein Nsp2 synthetic peptide polyclonal antibody Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly discloses an antigen peptide of a porcine reproductive and respiratory syndrome virus non-structural protein 2, a polyclonal antibody prepared by using the antigen peptide and application thereof. The amino acid sequence of the antigen peptide is shown as SEQ ID NO. 1. The invention also discloses a preparation method of the antigen peptide and a polyclonal antibody prepared by using the antigen peptide. The polyclonal antibody prepared by the invention has higher sensitivity and specificity, can specifically identify the Nsp2 protein, can be used for detecting the Nsp2 protein, identifying the protein function, locating cells and the like, provides an important experimental material for researching the biological function of the Nsp2 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 preparation and application of a PRRSV nonstructural protein Nsp2 synthetic peptide polyclonal antibody.
Background
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is a virulent infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). 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. At present, PRRSV is divided into two major genotypes PRRSV-1 and PRRSV-2 according to antigen differences, and PRRSV-2 type strains are mainly popular in China.
The PRRSV genome has the characteristic of high variation, and in 2006, china has outbreak high-pathogenicity PRRSV, and the strain partially lacks 30 amino acids in nonstructural protein 2 (Nonstructural proteins, nsp 2); new NADC34-like strain in China continuously lacks 100 amino acids in Nsp2 part. The reason for the high variation of PRRSV NSP2 gene is that the mutation of base, deletion of large fragment sequence and insertion of foreign gene or base sequence can be tolerated in the NSP2 hypervariable region. In view of the characteristics of PRRSV NSp2 gene variation, it is often used as a target gene for molecular epidemiological detection and research.
Nsp2 is a multi-domain protein with multiple functions, and has important biological functions in many aspects such as replication of PRRSV, regulation of host immune response, antiviral mechanism against host, variation and evolution, and in recent years, the function of Nsp2 and its molecular mechanism for exerting relevant functions have become the hot spot fields of research.
Chinese patent application CN116444682A discloses a biological protein degradation targeting chimeric for targeting and degrading PRRSV key replicase and application thereof. The biological protein degradation targeting chimeric is obtained by fusing 1 to a plurality of PRRSVnsp9 nanobody nsp9nb and/or nsp2 nanobody nsp2nb in series and then with a BTB structural domain in an E3 ubiquitin ligase joint protein SPOP. The biological protein degradation targeting chimeric body for targeting and degrading PRRSV key replicase is constructed by utilizing the screened nano antibody which specifically binds PRRSV key replicase NSp2 and NSp9 proteins and replacing an E3 ubiquitin ligase complex (SPOP) substrate recognition domain with the nano antibody, and the biological protein degradation targeting chimeric body does not relate to antigen peptide and polyclonal antibody.
The Chinese patent application CN116219068A designs a group of forward and reverse primers based on PRRSV NSP2 genes by comparing the sequences of PRRSV published sequences and the sequences of classical strains and highly pathogenic strains, designs two probes in a targeted manner according to the deletion characteristics of various strains, and designs an RT-RAA-LF detection method based on PRRSV NSP2 gene sequences by distinguishing the two strains specifically, which does not involve antigenic peptides and polyclonal antibodies.
Based on this, the present invention has been made. According to the invention, the antigen peptide prediction is carried out on PRRSV-NSp2, and the synthetic peptide antibody is prepared, so that the antibody can be effectively used for detecting PRRSV NSp2 proteins of different strains, and a powerful tool is provided for carrying out research on PRRSV NSp2 and prevention, control and diagnosis of PRRSV in the future.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention discloses an antigen peptide of a porcine reproductive and respiratory syndrome virus non-structural protein 2, a polyclonal antibody prepared by using the antigen peptide and application thereof. The amino acid sequence of the antigen peptide is shown as SEQ ID NO. 1. The invention also discloses a preparation method of the antigen peptide and a polyclonal antibody prepared by using the antigen peptide. The polyclonal antibody prepared by the invention has higher sensitivity and specificity, can specifically identify the Nsp2 protein, can be used for detecting the Nsp2 protein, identifying the protein function, locating cells and the like, provides an important experimental material for researching the biological function of the Nsp2 protein, and lays a biological foundation for detecting porcine reproductive and respiratory syndrome virus.
The invention realizes the technical effects by the following technical scheme:
the invention aims to provide a polyclonal antibody of porcine reproductive and respiratory syndrome virus Nsp2 protein and application thereof in detection of porcine reproductive and respiratory syndrome virus.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention claims an antigenic peptide of porcine reproductive and respiratory syndrome virus Nsp2, wherein the amino acid sequence of the antigenic peptide of porcine reproductive and respiratory syndrome virus Nsp2 is shown as SEQ ID NO. l.
The invention also claims a polyclonal antibody prepared by immunizing New Zealand white rabbits with the antigenic peptide of the porcine reproductive and respiratory syndrome virus Nsp 2.
The invention also claims a preparation method of the anti-porcine reproductive and respiratory syndrome virus Nsp2 polyclonal antibody, which comprises the following steps:
a) Preparation of synthetic peptide antigen: artificially synthesizing an amino acid sequence shown in SEQ ID No. l, and 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.
Preferably, in the preparation method of the anti-porcine reproductive and respiratory syndrome virus Nsp2 polyclonal antibody, the immunization mode in the step b) is subcutaneous injection or intramuscular injection, and the immunization amount of the antigen peptide is 500 mug each.
Preferably, in the preparation method of the anti-porcine reproductive and respiratory syndrome virus Nsp2 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, in the preparation method of the anti-porcine reproductive and respiratory syndrome virus Nsp2 polyclonal antibody, the step b) comprises the detection of antibody titer, and the detection of the titer and sensitivity of the antibody is carried out by an indirect ELISA method, wherein the titer of the polyclonal antibody is as high as 1:10 5 . The anti-PRRSV NSp2 polyclonal antibody was stored in PBS buffer containing 20% glycerol.
The use of said polyclonal antibody against Nsp2 of porcine reproductive and respiratory syndrome virus for Western Blot and indirect Immunofluorescence (IFA) detection of Nsp2 protein expression, preferably said polyclonal antibody is used for cell transient expression and expression detection of Nsp2 protein infected by different strains of virus, more preferably said polyclonal antibody is used for cell localization of Nsp 2.
The invention has the beneficial effects that: the Nsp2 polyclonal antibody meeting the requirement of subsequent experiments can be used for expression and cell location detection of Nsp2 protein, and detection of PRRSV infection samples of different strains, and provides an effective tool for researching the functions of PRRSV Nsp 2.
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.
Table 1 shows the antigen immune serum sensitivity detection by ELISA;
FIG. 1 is a graph showing the results of transiently expressed Flag-NSp2 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 different titers of HP-PRRSV infection of Marc-145 cells at the same time point;
FIG. 4 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 Nsp2
An antigenic peptide with an amino acid sequence of DQPAKDPRMSPRESD (comprising 1228 amino acids in total of NSp2 and amino acids at positions 817-831) as shown in SEQ ID NO. l is selected as the antigenic peptide of the polyclonal antibody, and the selection of the polypeptide sequence avoids the position of HP-PRRSV amino acid deletion. 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,diluting with anti-Nsp 2 polyclonal antibody at different concentrations, wherein the negative control wells are used for taking unimmunized rabbit serum as a control at 37 ℃ for 1h, wherein the concentrations are 1:3125, 1:6250, 1:12500, 1:25000, 1:50000 and 1:100000; after washing the plate, adding horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and incubating at 37 ℃ for 1h. After washing the plate, TMB was added to carry out a color reaction, and after the reaction was terminated, the absorbance at a wavelength of 450nm was measured. The potency assay results are shown in table 1: polyclonal antibodies have titers as high as 1:10 5 The titer can ensure that the polyclonal antibody can be used for detecting Nsp2 and ensure the quality of the antibody.
TABLE 1 ELISA titers detection of Nsp2 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 obtained Nsp2 protein synthetic peptide polyclonal antibody could be used in the subsequent Western Blot experiments, the resulting Nsp2 protein synthetic peptide polyclonal antibody was used to detect the over-expression of Flag14-Nsp2 plasmid in HEK 293T cells, by using a murine anti-Flag antibody and a rabbit anti-Nsp 2 polyclonal antibody as primary antibodies, fig. 1 is a graph of the results of the recombinant plasmid Flag14-Nsp2 expressed protein, as shown in fig. 1, both antibodies were able to show an over-expressed Nsp2 protein band and were consistent with the expected size. Therefore, the result shows that the prepared rabbit anti-NSP 2 polyclonal antibody can be used for detecting the NSP2 transiently expressed by cells.
EXAMPLE 4 HP-PRRSV verification of infection of Marc-145 cells at different time points at the same titer
Spreading Marc-145 cells with good state on a 60mm cell culture dish, incubating 0.1MOI HP-PRRSV (SH 01) and the cells for 2h, changing into 2% DMEM culture medium, continuously culturing, collecting cell samples after virus infection for 6h, 12h, 24h, 36h and 48h, and collecting uninfected cells for 6h and 48h as a control group. Protein expression was detected by using a rabbit anti-Nsp 2 polyclonal antibody, which can specifically detect a band of about 160kDa after 24h of virus infection, whose size was consistent with that predicted and increased as the virus infection time was prolonged, a rabbit anti-PRRSV N protein antibody and a mouse anti- β -actin antibody as primary antibodies, the detection results of which are shown in fig. 2. In addition, the samples tested were rechecked with antibodies against PRRSV N protein, which was detected about 12h after viral infection (fig. 2A). Further, the results of IFA detection showed that virus-infected cells could be specifically detected using rabbit anti-Nsp 2 polyclonal antibodies, 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 2 polyclonal antibody were identical to those of the anti-N protein (fig. 2B). Thus, these results demonstrate that the prepared rabbit anti-Nsp 2 polyclonal antibodies can be used for Nsp2 detection generated during viral infection.
Example 5 HP-verification of different titers of infected Marc-145 cells at the same time point
Marc-145 cells in good condition are spread on a 60mm cell culture dish, after the cells grow to about 70%, HP-PRRSV (SH 01) with the MOI of 0.1, 0.5 and 1 are respectively incubated with the cells for 2 hours and then replaced by a 2% DMEM medium for continuous culture, cell samples are collected 24 hours after virus infection, and uninfected cells are collected as a control group. By using a rabbit anti-Nsp 2 polyclonal antibody, a rabbit anti-PRRSV N protein antibody and a mouse anti- β -actin antibody as primary antibodies, the detection results are shown in fig. 3, and the rabbit anti-Nsp 2 polyclonal antibody can specifically detect Nsp2 protein expression in virus infection samples, and the Nsp2 protein amounts in the virus-receiving groups of 0.5MOI and 1MOI are both significantly increased compared to the virus-receiving group of 0.1MOI (fig. 3A). In addition, IFA results showed that the antigen peptide polyclonal antibodies were effective in detecting virus-infected cells, with a significant increase in the number of fluorescence-positive cells in the 0.5 and 1MOI virus-receiving groups compared to the 0.1MOI virus-receiving group (fig. 3B). These results further demonstrate that the prepared rabbit anti-Nsp 2 polyclonal antibodies can be used for detection of Nsp2 expression in HP-PRRSV infected samples.
EXAMPLE 6 verification of infection of Marc-145 cells with different strains of PRRSV
To verify whether the obtained NSP2 protein synthetic peptide polyclonal antibody can detect the NSP2 proteins of cells infected by different strains, a large amount of NSP2 proteins are expressed in the cells in view of 24 hours after virus infection. Thus, three strains SH01 and JX1R, CH R were selected to infect Marc-145 cells at a viral load of 1MOI, and samples were prepared and analyzed 24h after infection. The Western Blot results showed that NSp2 antigen peptide polyclonal antibodies specifically detected NSp2 protein expression in SH01 and JX1R virus infected samples, whereas NSp2 protein was not detected in CH1R virus infected samples, and N protein antibody detection results indicated that the virus infection was successful (FIG. 4A). In addition, the results of IFA showed that the antigen peptide polyclonal antibody can effectively detect virus-infected cells, and that the staining result of CH 1R-infected cells by Nsp2 antibody can overlap with the N protein detection image (fig. 4B). Therefore, these results further demonstrate that the prepared rabbit anti-Nsp 2 polyclonal antibody can be used for detecting the expression of Nsp2 in samples infected by HP-PRRSV and JX1R, but the antibody has weak reactivity to Nsp2 protein generated by CH1R infection.
Claims (9)
1. An antigen peptide of a non-structural protein 2 of the porcine reproductive and respiratory syndrome virus has an amino acid sequence shown in SEQ ID No. l.
2. The polyclonal antibody for resisting the porcine reproductive and respiratory syndrome virus nonstructural protein 2 is characterized in that the polyclonal antibody is prepared by taking a composite protein formed by crosslinking the antigen peptide of the porcine reproductive and respiratory syndrome virus nonstructural protein 2 modified at the C terminal and KLH carrier protein as an immunogen and immunizing animals.
3. A method for preparing the polyclonal antibody against the porcine reproductive and respiratory syndrome virus nonstructural protein 2 according to claim 2, comprising the steps of:
a) Preparation of synthetic peptide antigen: artificially synthesizing an amino acid sequence shown in SEQ ID No. l, and 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: purifying serum by Protein G affinity chromatography to obtain polyclonal antibody;
d) And (3) detecting the titer of the polyclonal antibody against the porcine reproductive and respiratory syndrome virus nonstructural protein 2 by an indirect ELISA method.
4. The method for preparing the polyclonal antibody against the nonstructural protein 2 of the porcine reproductive and respiratory syndrome virus according to claim 3, wherein the immunization mode in the step b) is subcutaneous injection or intramuscular injection, and the immunization amount of the antigen peptide is 500 mug each.
5. The method for preparing polyclonal antibody against non-structural protein 2 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.
6. The method for preparing the polyclonal antibody against the nonstructural protein 2 of the porcine reproductive and respiratory syndrome virus according to claim 3, wherein the titer and the sensitivity of the antibody are detected in the step d) by an indirect ELISA method, and the titer of the polyclonal antibody is as high as 1:10 5 The anti-porcine reproductive and respiratory syndrome virus nonstructural protein 2 polyclonal antibody was stored in 20% glycerol in PBS buffer.
7. Use of the polyclonal antibody against porcine reproductive and respiratory syndrome virus nonstructural protein 2 according to claim 2 for detection of porcine reproductive and respiratory syndrome virus nonstructural protein 2 protein expression.
8. The use of polyclonal antibodies against nonstructural protein 2 of porcine reproductive and respiratory syndrome virus according to claim 7, wherein said polyclonal antibodies are used for the detection of the expression of nonstructural protein 2 proteins of different strains.
9. The use of polyclonal antibody against the nonstructural protein 2 of porcine reproductive and respiratory syndrome virus according to claim 7, wherein said polyclonal antibody is used for protein function identification or cell localization of the nonstructural protein of porcine reproductive and respiratory syndrome virus.
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