A porcine reproductive and respiratory syndrome virus NADC30-like polypeptide SLA-I tetramer and its application

Abstract

This invention discloses a porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-like polypeptide SLA-I tetramer and its applications. The amino acid sequence of the porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer is shown in SEQ ID NO.2. The porcine PRRSV NADC30-like polypeptide SLA-I tetramer is formed by mixing a biotinylated porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer with FITC-streptavidin at a molar ratio of 4:1 during incubation. The tetramer pSLA-1-Tetramer of this invention can be quantitatively detected using flow cytometry to compare and evaluate the effects of different domestication strategies, providing a quantitative indicator for the domestication of porcine PRRSV.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to a porcine reproductive and respiratory syndrome virus NADC30-like polypeptide SLA- Tetramers and their applications. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease affecting reproduction and respiratory function in pigs, and is currently the most important underlying infectious disease threatening the pig industry. PRRSV was first reported isolated in my country in 1996. Over the past 20 years, the virus's highly mutable nature has led to increasingly complex clinical manifestations of the disease. Clinical infection results in abortion in sows, azoospermia in boars, and respiratory disorders in pigs of all ages. Biosecurity, vaccination, and herd isolation are the main economical and effective measures for controlling PRRS. Currently approved commercial vaccines are mostly attenuated live PRRS vaccines (MLV), including strains such as CH-1R, JXA1-R, HuN4-F112, TJM, and R98. MLV has been shown to provide good immunoprotection against homologous wild-type strains, but its cross-protection against heterologous strains is poor. Furthermore, the use of MLV has led to problems such as vaccine strain shedding, reversion to virulence, and recombination between wild-type and vaccine strains. The immunization strategy and protective effect, especially for piglets, remain highly controversial. Around 2013, a NADC30-like strain, genetically closely related to the US NADC30 strain, broke out and spread in my country, causing a "miscarriage storm" in pregnant sows. It has now become one of the dominant circulating PRRSV strains in my country, and existing vaccines offer very limited protection against NADC30-like strains. To address these issues, developing novel vaccines, such as peptide vaccines, is a hot research topic. These vaccines are composed of specific epitopes of the virus, resulting in a highly specific immune response and minimizing recombination in attenuated vaccines. However, vaccine development has a very long cycle.

[0003] Acclimation primarily refers to the domestication of replacement gilts. Its essence is to infect introduced pig herds with known PRRSV strains from the existing farm, bringing them to the same PRRSV infection level as the existing herd. Acclimation methods generally include three types: first, exposing replacement gilts to PRRSV-positive weaned piglets; second, injecting replacement gilts with PRRSV already present on the farm, which can be done using PRRSV-positive serum from the farm; and third, immunizing the herd with a live attenuated PRRSV vaccine. For large-scale farms, acclimation and quarantine management of the pig herd (especially replacement herds) after a PRRS outbreak are currently the main treatment methods. However, acclimation carries certain risks, mainly concerning strain safety, the timeliness of acclimation, strain selection, and serum quality. Therefore, there is an urgent need to establish objective and quantitative detection methods to determine the acclimation cycle and indicators for comparing and evaluating the actual effectiveness of different acclimation strategies.

[0004] MHC (Major histocompatibility complex)-peptide tetramer technology (pMHC-Tetramer) is a valuable technique for detecting specific T cells, such as those from pathogens or tumors. Four MHC-peptide complexes (pMHC) are assembled into pMHC-Tetramer using the biotin-protein ligase A (BirA)-streptavidin system, significantly enhancing the binding affinity of the MHC-peptide complex to the T cell receptor (TCR). Detection using flow cytometry offers high sensitivity and specificity. Current methods mostly involve preparing the SLA heavy and light chains separately, then refolding them in vitro with synthetic pathogen peptides to obtain the SLA heavy-light-peptide trimer (pSLA), and then constructing the tetramer (Tetramer pSLA) using streptavidin and cytosine. For immunosuppressive pathogens, such as PRRSV, cellular immunity plays a crucial role in antiviral infection. In cellular immune responses, cytotoxic T lymphocytes (CTLs) do not directly bind to antigen molecules or antigenic peptides. Instead, an effective immune response is formed by a ternary complex consisting of an antigen-specific TCR molecule, an MHC I molecule, and the corresponding specific antigenic peptide. Therefore, the binding of the TCR to MHC... -Using antigen peptide binding to quantify specific CTLs has become a new trend, solving the problem of extremely low affinity (~10) between monovalent MHC-peptide molecules and TCRs. -5 It has the drawbacks of being a high concentration of μmol / L and rapidly dissociating, resulting in a complex with a half-life of less than 1 min.

[0005] Swine leukocyte antigen (SLA) is the encoding product of the porcine major biocompatibility complex (MHC) gene, and is divided into SLA- The three main categories are SLA-II and SLA-III. Molecularly mediated cellular immune responses, classic SLA The gene-like structure includes three gene loci: SLA-1, SLA-2, and SLA-3. SLA- Gene-like proteins are glycoproteins composed of a heavy chain (α chain) and a light chain β2 microglobulin (β2m) linked nonvalently. Their α chain exhibits polymorphism, but β2m is singlet. SLA Heavy and light chains of viral molecules, along with viral peptides, can form complexes within cells, which are then presented to the cell surface and recognized by CD8+ T cell receptors, triggering a CTL immune response that helps the body clear the virus and resist disease. These functional viral peptides are potential vaccine candidate epitopes and can be used to develop peptide vaccines. In 2016, Gao Fengshan of Dalian University constructed pSLA-2-Tetramer for foot-and-mouth disease virus (FMDV) in pigs to screen and identify FMDV peptides. SLA-2 was expressed using the prokaryotic expression system pET-21a(+) / BL21, and inclusion bodies were extracted. FMDV peptides that elicit specific CTL responses were screened using enzyme-linked dot hybridization. The peptides were then renatured with the SLA-2 heavy chain and β2m light chain, biotinylated, and reacted with FITC-labeled streptavidin to generate pSLA-2-Tetramer. The function of Tetramer was then detected by flow cytometry, and Hu62 was identified as an FMDV-specific CTL epitope. In 2021, Xia Chun's team at China Agricultural University used a similar method to Gao Fengshan's to construct pSLA-1∗1502-Tetramer for the PRRSV attenuated vaccine strain (VR2332). pET-21a(+) / BL21 expressed the SLA-1 heavy chain and β2m light chain. Through a series of steps including denaturation, renaturation, and biotinylation of inclusion body proteins, followed by reaction with FITC-labeled streptavidin, pSLA-1∗1502-Tetramer was generated, identifying NSP9-TMP9 as a PRRSV CTL epitope. These studies demonstrate that Tetramer technology plays a crucial role in identifying virus-specific epitopes; however, it has not been practically used in large-scale farms. Whether it can evaluate the immunization and acclimatization effects of specific vaccines in pig herds, and provide a useful detection method for pig farming enterprises, remains unknown. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a monomer of a porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex.

[0007] Another object of the present invention is to provide a porcine PRRSV NADC30-like polypeptide SLA- Tetramer.

[0008] Another object of the present invention is to provide a porcine PRRSV NADC30-like polypeptide SLA- Applications of tetramers.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] A PRRSV NADC30-like specific polypeptide GP5, the amino acid sequence of which is shown in SEQ ID NO.1.

[0011] A monomer of a porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex is composed of the heavy chain α chain and light chain β2m of porcine leukocyte antigen SLA-1, the aforementioned PRRSV NADC30-like specific polypeptide GP5, and a linker (G4S)n in tandem. The fusion order of the heavy chain α chain and light chain β2m of porcine leukocyte antigen SLA-1, the PRRSV NADC30-like specific polypeptide GP5, and the linker (G4S)n is not fixed, and the number of repeats of the linker (G4S)n is n=1~7.

[0012] As a preferred embodiment of the present invention, the fusion sequence of the elements of the porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer is: GP5-linker(G4S)3-β2m-linker(G4S)4-α, and the amino acid sequence is shown in SEQ ID NO.2.

[0013] The coding gene for the porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer described in this invention has a preferred nucleotide sequence as shown in SEQ ID NO.3.

[0014] A porcine PRRSV NADC30-like polypeptide SLA- The tetramer is formed by incubating a mixture of biotinylated porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer and FITC-streptavidin at a molar ratio of 4:1.

[0015] A recombinant expression plasmid containing the encoding gene of the porcine PRRSV NADC30-like SLA-1 restriction CTL epitope complex monomer; preferably using pCold I as the initial vector.

[0016] A recombinant expression plasmid combination, characterized in that it comprises the recombinant expression plasmid described in this invention and a recombinant expression plasmid containing the full-length BirA gene; the full-length BirA gene is amplified by PCR using *Escherichia coli* DH5α genomic DNA as a template and primers SEQ ID NO.4 and SEQ ID NO.5; the initial plasmid of the recombinant expression plasmid containing the full-length BirA gene can be selected or modified as needed to differentiate it from pCold Ⅰ (Amp+) resistant prokaryotic expression plasmids, preferably the prokaryotic expression vector pET28a (Kan+). The porcine PRRSV NADC30-like polypeptide SLA- described in this invention... The method for preparing the tetramer includes the following steps:

[0017] (1) Preparation of biotinylated porcine PRRSV NADC30-like SLA-1 restriction CTL epitope complex monomer: The recombinant expression plasmid combination was co-transformed into Escherichia coli BL21(DE3) competent cells, and strains that could express two recombinant plasmids were screened on double-antibiotic (Amp+Kan+) LB plates; single colonies were picked and cultured at 37°C until OD600=0.4-1.0, IPTG was added to a final concentration of 0.1-1mM, and biotin was added to a final concentration of 10-100μM. Induction continued for 6h-24h, and the cells were lysed and purified by nickel column to obtain the biotinylated porcine PRRSV NADC30-like SLA-1 restriction CTL epitope complex monomer;

[0018] (2) Preparation of porcine PRRSV NADC30-like peptide SLA- Tetramer: The biotinylated porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer BGP5-SLA-1 prepared in the previous step was mixed with FITC-streptavidin at a molar ratio of 4:1 and incubated at room temperature in the dark for 30 min-1 h to form the tetramer BGP5-SLA-1-Tetramer.

[0019] The present invention describes the combination of porcine PRRSV NADC30-like SLA-1 restriction CTL epitope complex monomer, the encoding gene, and the recombinant expression plasmid in the preparation of porcine PRRSV NADC30-like polypeptide SLA-1. Applications in tetramers.

[0020] The porcine PRRSV NADC30-like polypeptide SLA- described in this invention Applications of tetramers in detecting cytotoxic lymphocytes, evaluating the immunization effect of PRRSV vaccines, or detecting cellular immune indicators during the PRRSV acclimatization process in pig herds.

[0021] A kit comprising: the porcine PRRSV NADC30-like polypeptide SLA- described in this invention. Tetramer.

[0022] As a preferred embodiment of the present invention, the kit comprises: the porcine PRRSV NADC30-like polypeptide SLA- Tetramer, PE-Mouse Anti-Pig CD8a monoclonal antibody / PerCP-Mouse Anti-Pig CD3a monoclonal antibody (BD Biosciences).

[0023] (1) In this invention, the dominant allele SLA-1*080101 heavy chain (α chain) and light chain (β2m) of commercial pigs, as well as the PRRSV NADC30-like specific polypeptide (GP5) sequence, were selected and cloned into the prokaryotic expression vector pColdⅠ(Amp+) carrying an ampicillin resistance expression cassette after codon optimization. The recombinant strain BL21(DE3) / pColdⅠ-gp5-β2m-α was constructed, and soluble expression of GP5-β2m-α was successfully achieved. This avoids the cumbersome steps of inclusion body washing, denaturation, and renaturation, and reduces excessive lipopolysaccharide contamination, significantly improving the polymer yield and thus reducing costs. In this invention, the fusion order between the three genes α chain, β2m, and gp5 is not fixed, and the number of Linker (G4S)n repeats is also not fixed. These are adjusted according to the expression effect, but all are within the scope of protection of this invention.

[0024] This invention utilizes the prokaryotic expression vector pET28a (Kan+) carrying a kanamycin resistance expression cassette. BirA is cloned into pET28a via homologous recombination to construct the recombinant expression plasmid pET28a-BirA, which is then stably expressed in the BL21(DE3) host bacterium. The prokaryotic expression plasmid used for cloning and expressing the BirA gene in this invention is not limited to pET28a; any plasmid selected or modified to differ from pCold Ⅰ (Amp+) resistance may be used, and all are within the scope of this invention.

[0025] This invention co-transforms the two plasmids pET28a(Kan+)-BirA and pCold(Amp+)-gp5-β2m-α, which can be successfully expressed individually, into *E. coli* BL21(DE3). After screening with double-antibiotic (Amp+Kan+) LB plates, recombinant bacteria BL21(DE3) / pET28a-BirA / pColdⅠ-gp5-β2m-α simultaneously expressing both plasmids were successfully obtained, achieving soluble expression and in vivo biotinylation of GP5-β2m-α in a single step. This simplifies the multiple steps reported in previous literature, including separate expression of the SLA heavy and light chains, denaturation and renaturation of inclusion body proteins, and in vitro biotinylation, into a single step, obtaining the biolabeled peptide monomer, BGP5-SLA-1, in one go. Then, FITC-labeled streptavidin is used to generate BGP5-SLA-1-Tetramer, significantly saving time and cost.

[0026] Beneficial effects:

[0027] This invention targets the currently dominant PRRSV NADC30-like strains. Through bioinformatics analysis, a group of antigenic peptides were obtained, which were identified as possessing good specificity and antigenicity. These peptides were then used to prepare pSLA-1-Tetramer, improving the protein form of SLA heavy and light chains expressed in vitro in *E. coli*, transforming it from inclusion bodies to soluble proteins, and converting in vitro biotinylation to in vivo biotinylation. This allows for the simultaneous completion of SLA expression and biotinylation, resulting in biotinylated pSLA and significantly improving the preparation efficiency of pSLA-Tetramer, greatly saving time and cost. pSLA-Tetramer significantly enhances its binding affinity to T-cell receptors, and a high proportion of cytotoxic lymphocytes can be detected using flow cytometry. It exhibits high sensitivity and specificity, enabling the evaluation of PRRSV vaccine efficacy and the detection of cellular immune indicators during PRRSV acclimatization in pig herds. Attached Figure Description

[0028] Figure 1 The recognition frequency of the four epitope peptides in 10 infected pigs.

[0029] Figure 2 Statistical graph of IFN-γ secretion in porcine T cells induced by PRRSV infection from the SEQ ID.1 epitope peptide detected by Elispot.

[0030] Figure 3 The recombinant plasmid pCold I-gp5-β2m-α was identified by NdeI and XhoI restriction enzyme digestion. M: 1kB molecular weight standard; 1: empty pCold I plasmid; 2: recombinant plasmid digested with NdeI and XhoI.

[0031] Figure 4SDS-PAGE analysis was performed to determine the expression of the recombinant protein gp5-β2m-α. M: molecular weight standard in pre-stained samples; 1: empty vector BL21 / pCold 1: Whole cell lysate after induction; 2: Empty vector BL21 / pCold 3: Supernatant of whole-cell lysate after centrifugation following induction; BL21 / pCold empty vector 4: Recombinant bacteria BL21 / pCold lysate after centrifugation; 5: Recombinant BL21 / pCold whole bacterial lysate after induction with -gp5-β2m-α; 6: Recombinant BL21 / pCold #imgpt22# -gp5-β2m-α induced whole bacterial lysate after centrifugation and precipitate.

[0032] Figure 5 Recombinant plasmid pET28-BirA was identified by enzyme digestion. M: 1 kB molecular weight standard; 1-4: Recombinant plasmid pET28-BirA digested with NcoI and XhoI.

[0033] Figure 6 SDS-PAGE analysis of recombinant protein BirA expression. M: molecular weight standard in pre-stained samples;

[0034] 1: Whole-cell lysate induced by empty vector BL21 / pET28a; 2: Supernatant of whole-cell lysate induced by empty vector BL21 / pET28a-BirA after centrifugation; 3: Precipitate of whole-cell lysate induced by empty vector BL21 / pET28a-BirA after centrifugation.

[0035] Figure 7 SDS-PAGE analysis was performed to determine the purity of recombinant protein BGP5-SLA-1. M: molecular weight standard in pre-stained protein; 1-2: purified BGP5-SLA-1.

[0036] Figure 8 Western blot analysis was used to determine the biotinylation status of BGP5-SLA-1. M: molecular weight standard in pre-stained samples; 1-2: unbiotinylated GP5-SLA-1; 3-4: biotinylated BGP5-SLA-1.

[0037] Figure 9The proportion of specific T cells in peripheral blood lymphocytes of PRRSV NADC30-like challenged pigs was analyzed using BGP5-SLA-1-Tetramer (flow cytometry). In the diagram: A and B represent tetramer-stained PBMCs from unchallenged pigs, with CD8+CTL+ cell proportions of 4.9 and 4.7, respectively; CF represent tetramer-stained PBMCs from four challenged pigs, with CD8+CTL+ cell proportions of 8.67, 10.9, 8.21, and 11, respectively.

[0038] Figure 10 The proportion of specific T cells in peripheral blood lymphocytes of PRRSV NADC30-like challenged pigs was analyzed using BGP5-SLA-1-Tetramer (statistical graph). In the graph: controls represent tetramer-stained, non-challenged pig PBMCs; infected pigs represent tetramer-stained, four challenged pig PBMCs. Detailed Implementation

[0039] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0041] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0042] Example 1: Design and synthesis of NADC30-like SLA-1-restricted CTL epitopes in porcine PRRSV

[0043] Submit the SLA-1* 080101 allele, which appears frequently in the pig herd, online. www.cbs.dtu.dk / services / NetMHCpan / ), and then input the sequences of 7 proteins (GP2a, GP3, GP4, GP5, M, E, and N) of the PRRSV NADC30-like strain (the strain CZ2020 isolated at the test site) into the prediction website to analyze their affinity with SLA-1* 080101. Based on the affinity scores, 4 epitope peptides were screened and synthesized, among which SEQ ID NO.1 (GP5): LLDTKGKLY had the highest affinity, as shown in Table 1. All peptides were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0044] Table 1: Peptide sequence information in this invention

[0045] N LSDSGRISY GP5 LLDTKGKLY (SEQ ID NO.1) GP4 HSNPPSATF GP3 ILEPGRSLW

[0046] Example 2: ELISpot method was used to detect the ability of the four epitope peptides in Example 1 to induce T cells to secrete IFN-γ.

[0047] PBMC cell isolation: Infected porcine peripheral blood lymphocytes (PBMCs) were isolated using the density gradient method. 5 mL of collected peripheral blood was diluted 1:1 with sample diluent, and the diluted peripheral blood was slowly spread on top of an equal volume of lymphocyte separation medium. The mixture was centrifuged at 250 g / min for 10 min. White membrane cells were carefully aspirated into a new centrifuge tube using a pipette. The cells were washed with 5-10 times their volume of washing buffer and centrifuged at 250 g / min for 10 min. The washing was repeated twice, and the cells were resuspended in complete culture medium containing 10% FBS for later use.

[0048] ELISpot assay: After obtaining the PBMC cell count as described above, the count was quantified to a ratio of 1×10⁻⁶. 6 Cells / mL; 200 μL of cell suspension was added to each well of an ELISpot plate, for a total of 12 wells. The four candidate peptides shown in Table 1 were added to eight wells at a concentration of 10 μg / mL, with two replicates for each peptide; 10 μg / mL concanavalin A (ConA) was added to two positive control wells, and complete culture medium without the peptide was added to two negative control wells (NC); the ELISpot plate was incubated at 37°C with 5% CO2 for 24 h. The ELISpot plate was then removed, washed three times with washing buffer, blotted dry, and 100 μL of enzyme-labeled anti-IFN-γ antibody (Wuhan Boster Biological Engineering Co., Ltd.) was added, incubated for 2 h, washed three times with washing buffer, blotted dry, and 100 μL of chromogenic reagent was added. The reaction was stopped with ultrapure water after obvious spots appeared in the positive control wells; the plate was dried, read, counted, and corrected. Specific T cell frequency was measured using 10... 6 Spot-forming cells (SFC / 10) in PBMCs 6 It is expressed as ) . Criteria for judging a positive reaction: ①SFC / 10 6 >50; ② The number of spots in the peptide-stimulated wells is greater than or equal to twice that in the negative control wells. If both ① and ② are met, the reaction is considered positive. Ten pig PBMCs were stimulated with four candidate epitope peptides, and the recognition frequency of the candidate epitope peptides in the ten pigs was counted. Peptide recognition rate = (number of positive reactions / total number of tests) × 100%, as shown in the figure. Figure 1 As shown. From Figure 1As can be seen, all four epitope peptides of this invention can be recognized by pigs infected with SLA-1* 080101 PRRSV NADC30-like. The GP5 polypeptide (LLDTKGKLY, SEQ ID NO.1) epitope peptide induces the secretion of IFN-γ by cells of pigs infected with SLA-1* 080101 PRRSV NADC30-like, as shown in the results. Figure 2 As shown. Combined with Figure 1 and Figure 2 It is evident that the GP5 peptide (LLDTKGKLY, SEQ ID NO.1) can act as an epitope peptide to specifically stimulate the secretion of IFN-γ by PBMCs in SLA-1* 080101 PRRSV NADC30-like infected pigs.

[0049] Example 3 Construction of biotinylated SLA-1 complex monomer (BpSLA-1)

[0050] 3.1 Construction of SLA-1 complex monomeric recombinant bacteria

[0051] First, the nucleotide sequences of the SLA-1*080101 allele α chain, β2m, and GP5 polypeptide (SEQ ID NO.1) were optimized according to the E. coli preferred codons. A BirA substrate peptide sequence (BSP) was added to the C-terminus of the α chain, and a linker (G4S)3 was inserted between the α chain, β2m, and SEQ ID NO.1. Then, the plasmid was cloned into the prokaryotic expression plasmid pCold I using restriction endonucleases NdeI and XhoI to construct the recombinant plasmid pCold I-gp5-β2m-α, which was transformed into BL21(DE3) competent cells. Single colonies were picked from the plate, and the plasmid was extracted and identified by digestion with restriction endonucleases NdeI and XhoI. A 1300 bp target fragment was visible, indicating successful acquisition of the positive recombinant bacterium BL21 / pCold I-gp5-β2m-α. Figure 3 The fusion order among the three genes α, β2m, and gp5 is not fixed, nor is the number of repeats of Linker (G4S)n. These are adjusted based on expression efficacy, but all fall within the scope of this invention. In this invention, the preferred tandem sequence is “GP5-linker(G4S)3-β2m-linker(G4S)4-α”, as shown in SEQ ID NO.2, with NdeI / XhoI restriction sites added at both ends, resulting in the nucleotide sequence shown in SEQ ID NO.3.

[0052] 3.2 Expression of SLA-1 complex monomeric recombinant bacteria

[0053] The above-identified positive recombinant bacteria were inoculated into 5 mL of medium containing ampicillin (Amp+) and cultured at 37°C until OD600 = 0.6. IPTG was then added to a final concentration of 0.1 mM, and induction continued at 16°C for 20 h. After lysis, 0.5 mL of bacterial cell pellet was collected and subjected to SDS-PAGE to detect protein expression. The recombinant bacteria BL21(DE3) / pCold I-gp5-β2m-α showed a clear protein band at approximately 45 kDa after induction. Figure 4 .

[0054] 3.3 Cloning and expression of the biotin ligase (BirA) gene

[0055] Using *Escherichia coli* DH5α as a template strain, 1 mL of overnight culture was boiled to prepare *E. coli* genomic DNA. A pair of homologous recombination primers (Bity-F: 5-ttaagaaggagatataCCATGGacaccgtgccactgaaattg-3 (SEQ ID NO.4); Bity-R: 5-cagtggtggtggtggtggCTCGAGtttctgcactacgcag-3 (SEQ ID NO.5)) were designed based on the GenBank accession sequence (Gene ID: 948469) to amplify the full-length BirA gene. The ligation of pET28a was performed by digestion with NcoI and XhoI using homologous recombinase (referring to the Novitane homologous recombinase instructions), followed by transformation into *E. coli* DH5α competent cells to obtain the recombinant strain DH5α / pET28a-BirA. Figure 5 .

[0056] The positive plasmid pET28a-BirA was then transformed into competent E. coli BL21(DE3) cells. The next day, single colonies were picked and transferred to LB (Kan+) medium for further culture, followed by transfer to fresh LB (Kan+) medium. The cells were cultured at 37°C until OD600 = 0.5, at which point IPTG was added to a final concentration of 0.1 mM, and induction continued for 12 h. The bacterial pellet was then lysed and subjected to SDS-PAGE to detect protein expression. The recombinant BL21(DE3) / pET28a-BirA showed a clear protein band at approximately 33 kDa after induction. Figure 6 .

[0057] 3.4 Co-transformation and SLA-1 complex monomeric expression

[0058] Simultaneously, two plasmids, pET28a-BirA and pCold I-gp5-β2m-α, were co-transformed into E. coli BL21(DE3) competent cells. Strains expressing both recombinant plasmids (BL21(DE3) / pET28a-BirA / pCold I-gp5-β2m-α) were selected using double-antibiotic (Amp+Kan+) LB agar plates. The next day, single colonies were picked and transferred to LB medium for further culture. The next day, the cells were transferred to fresh LB medium and cultured at 37°C until OD600 = 0.6. IPTG was added to a final concentration of 0.1 mM, and biotin was added to a final concentration of 20 μM. Induction was continued for 12 h. Cell lysis was then performed using SDS-PAGE to detect protein expression. After induction, the recombinant strain BL21(DE3) / pET28a-BirA / pCold I-gp5-β2m-α showed a biotinylated target protein band at a position slightly below 45kD, namely Bio-GP5-β2m-α (hereinafter referred to as BGP5-SLA-1).

[0059] 3.5 Purification and Identification of BGP5-SLA-1 Complex Monomers

[0060] Purification was performed using the Bio-Rad NGC protein purifier according to the Cytiva Histrip nickel column instruction manual. The main steps were as follows: Large batches of induced recombinant protein were aliquoted into 50mL centrifuge tubes, centrifuged at 12000rpm for 30min at 4℃, filtered through a 0.22μm filter to remove impurities, and temporarily stored at low temperature for later use. Then, the protein purifier was turned on, and the AB tubing was washed with deionized water to remove 20% anhydrous ethanol from the system. The nickel column was then loaded while the liquid was flowing, avoiding air intake, and the flow rate was set to 1mL / min. The new column was then washed sequentially with ddH2O and binding buffer until the UV 280nm line was horizontal. The protein sample was then loaded, and the peak time at UV 280nm was closely monitored. The flow-through was collected in a centrifuge tube, and a certain volume of binding buffer was used to remove impurities until the UV 280nm line was horizontal. Afterward, the protein sample was eluted with elution buffer; if a peak was observed, it was collected, and the collected sample was placed on ice until the UV 280nm line was horizontal. To further improve protein purity, the Elution-eluted protein was re-coated onto a nickel column, and the above steps were repeated for secondary purification. Finally, the collected target protein was sampled and analyzed by SDS-PAGE. Figure 7To determine whether the purified target protein BGP5-SLA-1 was biotinylated, Western blot analysis was performed using a semi-dry transfer instrument after SDS-PAGE. HRP-chain and cytosine were used as detection antibodies. Compared to the unbiotinylated GP5-SLA-1 complex monomer control, the HRP-chain and cytosine-labeled proteins showed distinct blot bands in the lanes. Figure 8 .

[0061] Example 4: BGP5-SLA-1 tetramer detection of PRRSV-specific T cells

[0062] 4.1 Construction of BGP5-SLA-1 tetramer

[0063] The above-mentioned biotinylated monomer BGP5-SLA-1 was mixed with FITC-streptavidin at a molar ratio of 4:1 and incubated at room temperature in the dark for 30 min to 1 h to form a tetramer, namely BGP5-SLA-1-Tetramer.

[0064] 4.2 BGP5-SLA-1-Tetramer Flow Cytometry

[0065] Six 1-month-old Duroc boars (4 for the experiment, 2 for the control) were selected and were negative for both PRRSV antibodies and antigens. The nasal cavity was used to challenge each boar with 2 mL of PRRSV NADC30-like CZ2020, while the control boar was challenged with 2 mL of cell culture medium. Fresh anticoagulated blood was collected from both the challenged and control boars 21 days post-challenge. The procedure was performed according to the instructions of the porcine peripheral blood lymphocyte separation kit. The main steps included: taking 3-5 mL of fresh anticoagulated blood, mixing it with a 1:1 ratio of diluent, centrifuging, carefully aspirating the separation medium and lymphocyte layer into a new centrifuge tube using a pipette, washing repeatedly, and finally resuspending the cell pellet in an appropriate volume of FACS washing buffer (containing 0.1% sodium azide and 0.1% BSA in a 1:1 PBS buffer). Add 5 μL of BpSLA-1-Tetramer and 2.5 μg (5 μL) of PE-anti-pig CD8a antibody to the cell suspension, incubate at 4°C in the dark for 30-60 min, then centrifuge to harvest the cell pellet, wash twice with 1 mL of FACS, resuspend the cell pellet in an appropriate amount of FACS washing buffer, and analyze. Double staining of porcine PBMCs with PE-Anti-pig CD8a and BpSLA-1-Tetramer showed that the proportion of CTLs specific to the GP5 peptide epitope was significantly higher than that of control pigs. Figure 9 and Figure 10 The results showed that BpSLA-1-Tetramer was successfully prepared and could be used to evaluate the immunogenicity of PRRSV NADC 30-liker infection in pigs.

Claims

1. A monomer of a porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex, characterized in that, Composed of the heavy chain α chain and light chain of porcine leukocyte antigen SLA-1 β2m The complex consists of a PRRSV NADC30-like specific polypeptide GP5 with the amino acid sequence shown in SEQ ID NO.1, and a Linker (G4S)n tandemly connected. The fusion sequence of the elements of the porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer is: GP5-linker(G4S)3- β2m - linker(G4S)4-α, amino acid sequence as shown in SEQ ID NO.

2.

2. The gene encoding the monomer of the porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

3.

4. A porcine PRRSV NADC30-like polypeptide SLA-I tetramer, characterized in that, The tetramer is formed by incubating a mixture of biotinylated porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomers of claim 1 and FITC-streptavidin at a molar ratio of 4:

1.

5. A recombinant expression plasmid, characterized in that, The encoding gene containing the monomer of the porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex as described in claim 2 or 3.

6. The recombinant expression plasmid according to claim 5, characterized in that, pCold I was used as the initial vector.

7. A recombinant expression plasmid combination, characterized in that, It consists of the recombinant expression plasmid as described in claim 5 and a recombinant expression plasmid containing the full-length BirA gene; the full-length BirA gene is obtained by PCR amplification using Escherichia coli DH5a genomic DNA as a template and primers SEQ ID NO.4 and SEQ ID NO.5; the initial plasmid of the recombinant expression plasmid containing the full-length BirA gene is selected or modified as needed to distinguish it from the prokaryotic expression plasmid resistant to pCold Ⅰ (Amp+).

8. The recombinant expression plasmid combination according to claim 7, characterized in that, The prokaryotic expression plasmid is pET28a (Kan+).

9. The method for preparing the porcine PRRSV NADC30-like polypeptide SLA-I tetramer according to claim 4, characterized in that, Includes the following steps: (1) Preparation of biotinylated porcine PRRSV NADC30-like SLA-1 restriction CTL epitope complex monomer: The recombinant expression plasmid combination described in claim 7 was co-transformed into Escherichia coli BL21(DE3) competent cells. Strains that could express two recombinant plasmids were screened on Amp+Kan+double antibiotic LB plates. Single colonies were picked and cultured at 37°C until OD600=0.4-1.

0. IPTG was added to a final concentration of 0.1-1mM, and biotin was added to a final concentration of 10-100μM. Induction was continued for 6h-24h. After cell lysis, the biotinylated porcine PRRSV NADC30-like SLA-1 restriction CTL epitope complex monomer was obtained by nickel column purification. (2) Preparation of porcine PRRSV NADC30-like polypeptide SLA-I tetramer: The biotinylated porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer BGP5-SLA-1 prepared in the previous step was mixed with FITC-streptavidin at a molar ratio of 4:1 and incubated at room temperature in the dark for 30 min-1 h to form tetramer BGP5-SLA-1-Tetramer.

10. The use of the porcine PRRSV NADC30-like SLA-1 restricted CTL epitope complex monomer of claim 1, the encoding gene of claim 2, and the recombinant expression plasmid combination of claim 7 in the preparation of the porcine PRRSV NADC30-like polypeptide SLA-I tetramer of claim 4.

11. A reagent kit, characterized in that... include: The porcine PRRSV NADC30-like polypeptide SLA-I tetramer as described in claim 4.

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