Porcine reproductive and respiratory syndrome virus attenuated strain, identification kit as well as preparation method and application of porcine reproductive and respiratory syndrome virus attenuated strain

Abstract

The invention provides a porcine reproductive and respiratory syndrome virus attenuated strain, an identification kit and a preparation method and application of the porcine reproductive and respiratory syndrome virus attenuated strain, and belongs to the technical field of virus prevention and treatment. The invention aims to provide a porcine reproductive and respiratory syndrome virus recombinant attenuated vaccine strain, and the vaccine strain can effectively distinguish wild strain infection and vaccine strain immunization. The invention provides an attenuated strain H18-R of a porcine reproductive and respiratory syndrome virus, the preservation number of the attenuated strain H18-R of the porcine reproductive and respiratory syndrome virus is CGMCC (China General Microbiological Culture Collection Center) NO.46203, and the preservation date is October 17, 2024. After immunization, the H18-R vaccine is safe to pigs, has no pathogenicity, can effectively induce an organism to generate immune response, resists the attack of porcine reproductive and respiratory syndrome virus, and provides good protection for immunized pigs.

Description

Technical Field

[0001] The invention belongs to the technical field of virus prevention and treatment, and specifically relates to an attenuated strain of porcine reproductive and respiratory syndrome virus, an identification kit, and a preparation method and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease that seriously harms the world's pig industry. It is commonly known as "blue ear disease". The disease is mainly characterized by reproductive disorders in pregnant sows and respiratory symptoms in piglets. Porcine reproductive and respiratory syndrome virus is classified as a member of the Nidovirales order, Arteriviridae family, and Betaarterivirus genus. It is further divided into Betaarterivirus suid 1 (formerly European type) and Betaarterivirus suid 2 (formerly North American type). PRRSV is a single-stranded positive-sense RNA virus with a genome length of about 15 kb. The upstream of the 5' non-coding region is a cap structure, and the downstream of the 3' non-coding region is a Poly (A) tail. Its genome encodes at least 11 open reading frames (ORFs): ORF1a, ORF1b, ORF2a, ORF2b, ORF3-7, ORF5a and NSP2 (TF). ORF1 accounts for the largest proportion of the viral genome (about 80%), which encodes the replicase complex, which is translated into polyproteins pp1a and pp1ab through the ribosomal frameshift mechanism. The three proteases encoded by ORF1a cleave at least 16 nonstructural proteins (NSP), among which NSP2 has the greatest degree of variation. In 1987, the disease was first discovered in American pigs and then spread throughout the world. In 1996, Guo Baoqing and others isolated PRRSV for the first time in China. Since then, PRRSV has been prevalent in my country, and has continued to mutate and recombine, causing multiple rounds of PRRS outbreaks. At present, the prevalence of PRRSV in my country has mainly experienced three stages. The first stage is mainly the classical strain stage represented by the CH-1a strain; the second stage is mainly the highly pathogenic PRRSV (HP-PRRSV) epidemic stage represented by the JXA1, HuN4, and TJ strains; the third stage is mainly the NADC30-like PRRSV epidemic stage. Since 2015, L1 lineage-type NADC30PRRSV has gradually become the dominant strain in my country.

[0003] Vaccine immunization is an effective means of preventing and controlling PRRS. Domestic commercial vaccines for PRRSV mainly include inactivated vaccines, live attenuated vaccines and genetically engineered vaccines, which are mainly targeted at classical strains and HP-PRRSV. These vaccines have played a positive role in preventing PRRSV, but as the virus continues to mutate, the existing commercial vaccine strains cannot provide complete immune protection against NADC30-like PRRSV, so more effective vaccine strains need to be developed. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant attenuated vaccine strain of porcine reproductive and respiratory syndrome virus, and the vaccine strain can effectively distinguish between wild strain infection and vaccine strain immunity.

[0005] The present invention provides an attenuated strain H18-R of arterivirus porcine reproductive and respiratory syndrome virus (PRRSV), which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC NO.46203 and a deposit date of October 17, 2024.

[0006] The present invention provides a porcine reproductive and respiratory syndrome virus vaccine, wherein the vaccine comprises the attenuated strain H18-R of the porcine reproductive and respiratory syndrome virus according to claim 1.

[0007] It is further defined that the vaccine also includes a pharmaceutical carrier or a pharmaceutically acceptable excipient.

[0008] The present invention provides an application of the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus in the preparation of a vaccine or medicine for preventing blue ear disease in pigs.

[0009] The invention provides an ELISA detection kit for detecting porcine reproductive and respiratory syndrome virus and identifying and distinguishing the porcine reproductive and respiratory syndrome virus attenuated strain H18-R. The kit contains the characteristic deletion protein of the porcine reproductive and respiratory syndrome virus attenuated strain H18-R, and the amino acid sequence of the protein is SEQ ID NO.2.

[0010] Further defined, the kit comprises a first antibody and a second antibody, wherein the first antibody comprises PRRS positive serum, and the second antibody is HRP-labeled goat anti-swine IgG (H+L).

[0011] The present invention provides the use of the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus, a protein with an amino acid sequence as shown in SEQ ID NO.2, a microbial cell carrying the protein as shown in SEQ ID NO.2, a coding gene SEQ ID NO.2 encoding the protein as shown in SEQ ID NO.2, and a recombinant vector containing the coding gene SEQ ID NO.1 in the preparation of a diagnostic and detection kit for porcine reproductive and respiratory syndrome virus.

[0012] The present invention provides an antigenic peptide of RRS virus, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] The present invention provides a nucleotide encoding the above-mentioned antigen peptide.

[0014] The present invention provides a recombinant vector or recombinant microbial cell containing the above nucleotides, which is used in preparing an ELISA detection kit for detecting porcine reproductive and respiratory syndrome virus and identifying and distinguishing the porcine reproductive and respiratory syndrome virus attenuated strain H18-R.

[0015] Beneficial effects: In one aspect of the present invention, a recombinant attenuated vaccine strain of porcine reproductive and respiratory syndrome is provided, the parent strain of the recombinant attenuated vaccine strain is a naturally recombinant porcine reproductive and respiratory syndrome virus, and the structural proteins with antigenic function are respectively from lineage 1 and lineage 8 strains, and are domesticated into attenuated vaccine strain H18-R through artificial continuous passage. The H18-R vaccine is safe and non-pathogenic to pigs after immunization, and can effectively induce the body to produce an immune response to resist the attack of porcine reproductive and respiratory syndrome virus, providing good protection for immunized pigs.

[0016] On the other hand, the present invention also provides a serological differential diagnosis method for distinguishing the above-mentioned porcine reproductive and respiratory syndrome recombinant attenuated vaccine strain from the naturally infected wild strain. In the past, it was difficult to distinguish between vaccine-immunized pigs and wild-virus naturally infected pigs when the attenuated vaccine strains were used to inoculate pigs. Therefore, it is very important to establish a differential diagnosis method that can distinguish between vaccine-immunized pigs and wild-virus infected pigs in terms of serology or etiology. According to the unique deletion characteristics of the attenuated vaccine strain H18-R NSP2, the present invention uses the 169 amino acids missing from the prokaryotic expression of the NSP2 protein as the coating antigen to establish an ELISA antibody detection method. The method has a negative test result for the serum of pigs immunized with the H18-R strain, a positive test result for the serum of pigs infected with the parental strain, the serum of pigs infected with the wild strain of PRRSV, and the positive serum of pigs immunized with the commercial PRRS vaccine, and has no cross reaction with the positive serum of other pathogens (ASFV, CSFV, PRV, PEDV, PPV and PCV-2). This ELISA method has good specificity and sensitivity, and can effectively distinguish pigs naturally infected with wild strains from pigs immunized with vaccine strains, meeting the clinical needs for differential diagnosis between vaccine-immunized strains and natural wild strains.

[0017] [Biological preservation information]: A attenuated strain H18-R of porcine reproductive and respiratory syndrome virus (PRRSV). The attenuated strain H18-R of porcine reproductive and respiratory syndrome virus is classified and named Arterivirus, with the preservation number CGMCC NO.46203. The preservation date is October 17, 2024, General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Recombination analysis of the genome sequence of the attenuated vaccine strain H18-R;

[0019] Figure 2 The level of PRRSV N protein antibody in the serum of piglets immunized with the attenuated vaccine strain H18-R;

[0020] Figure 3 Clinical symptom scores of piglets after immunization with attenuated vaccine strain H18-R;

[0021] Figure 4 Changes in body temperature of piglets after immunization with attenuated vaccine strain H18-R;

[0022] Figure 5 Serum viral load of piglets after immunization with attenuated vaccine strain H18-R;

[0023] Figure 6 Expression, purification and identification of the characteristic missing protein D169 of the attenuated vaccine strain H18-R. Lane M is the protein molecular mass standard; (a): 1: pET-24a, empty control, induced expression, whole bacterial solution; 2: pET-24a-D169, uninduced expression, whole bacterial solution; 3: pET-24a-D169, induced expression, whole bacterial solution; 4: pET-24a-D169, induced expression, supernatant; 5: pET-24a-D169, induced expression, precipitation; (b): 1: SDS-PAGE verification of purified D169; (c): 1: Western Blot verification of purified D169 (His tag antibody, 1:10000 dilution); (d): 1: Western Blot verification of purified D169 (PRRS positive serum, 1:100 dilution);

[0024] Figure 7 Test results of ELISA kit based on D169 protein; (a) specificity test; (b) sensitivity test;

[0025] Figure 8 The ELISA kit based on D169 protein was used to perform differential diagnosis on PRRSV positive sera from different sources; (a) positive sera from pigs infected with the parental strain of the recombinant attenuated vaccine; (b) positive sera from pigs immunized with the current domestic commercial attenuated live vaccine; (c) positive sera from pigs infected with clinical wild virus; (d) sera from pigs immunized with the attenuated vaccine strain H18-R (PRRSV N protein antibody positive detected by IDEXX PRRSVX3). DETAILED DESCRIPTION

[0026] Example 1. Domestication and identification of PRRSV attenuated vaccine strain H18-R

[0027] 1. Materials and Methods

[0028] 1.1 Virus strains and cells

[0029] PRRSV H18 strain (full name HeB-108 strain, GenBank accession no. MN046224.1) was isolated and preserved by our laboratory. Monkey embryonic kidney epithelial cells Marc-145 were also preserved by our laboratory.

[0030] 1.2 Serial passage and domestication of PRRSV H18 strain

[0031] Marc-145 cells were inoculated into T-25 cell flasks. When the cell confluence reached 80%, the culture medium was replaced with 2% FBS DMEM cell maintenance medium. 500 μL of virus cell culture supernatant was taken, thawed at room temperature, centrifuged at 2000rpm for 2min to remove cell debris, inoculated into Marc-145 cells, and cultured in a 37°C, 5% CO2 cell culture incubator. After 2 days of virus inoculation, the cells and supernatant were collected and frozen and thawed once at -80°C. The harvested cell supernatant was used as the parent virus to infect Marc-145 cells again, and the virus was continuously subcultured on Marc-145 cells to the 130th generation. The 130th generation attenuated strain was named H18-R.

[0032] 1.3 PRRSV H18-R whole genome amplification and sequencing

[0033] Amplification primers were designed using Oligo 6.0 to cover the entire viral genome (Table 1). The entire genome was amplified in 12 fragments, each about 1.4 kb in size, with 100 bp of overlap between the fragments.

[0034] Take 140 μL H18 cell supernatant and use the virus extraction kit QIAamp Viral RNA Mini Kit to extract viral RNA. Refer to the instructions for specific operations. TM II 1st Strand cDNA synthesis Kit was used to synthesize the first strand of cDNA. The specific process is as follows: 1 μL of Random 6mers (50 μM), 1 μL of dNTP Mixture (10 mM each), 8 μL of template RNA, the above reaction mixture was kept at 65°C, and then quickly cooled on ice. Then, 4 μL of 5× PrimeScript II Buffer, 1 μL of RNases Inhibitor (40 U / μL), 1 μL of PrimeScript II RTase (200 U / μL) were added to the above denatured reaction solution, and 20 μL was made up with RNase Free dH2O. The reaction conditions were 30°C for 5 min, 42°C for 1 h, and 70°C for 15 min.

[0035] Using cDNA as a template, high-fidelity DNA polymerase was used to amplify each target fragment. The amplification system was: 10 μL of 5×Q5 reaction buffer, 1 μL of 10 mM dNTPs, 2.5 μL of 10 μM forward and reverse primers, 0.5 μL of Q5 ultra-fidelity DNA polymerase, 2 μL of template cDNA, and 50 μL of ddH2O to complete the system. The reaction conditions were: 98°C for 30 s; 98°C for 10 s, 53°C to 60°C for 30 s, 72°C for 1 min and 30 s, amplification for 30 cycles; 72°C for 2 min, and stored at 4°C. The amplification product was observed by 1% agarose gel electrophoresis. After gel recovery and purification, the target fragment was sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. The sequencing results were compared by multiple sequence alignment using Lasergene software, and the viral sequence was determined in combination with the sequencing peak graph.

[0036] Table 1 Primers for viral whole genome amplification

[0037]

[0038]

[0039] 1.4 Recombination analysis

[0040] Representative strains of each branch of PRRSV were selected as reference strains, including L1 branch NADC30 (GenBank accession no.MH500776), L2 branch XW008 (GenBank accession no.KF724404), L3 branch MD001 (GenBank accession no.KP998431), L4 branch EDRD-1 (GenBank accession no.AB288356), L5 branch VR-2332 (GenBank accession no.AY150564), L6 branch P129 (GenBank accessionno.AF494042), L7 branch SP (GenBank accession no.AF184212), L8 branch JXA1 (GenBank accession no.EF112445) and L9 branch MN30100 (GenBank accession no.EF536000). After the reference sequence and the target sequence were aligned by MEGA 7, Simplot (v3.5.1) was used for similarity mapping to determine the recombination signal and recombination position.

[0041] 2. Results

[0042] H18 was inoculated with Marc-145 cells, and after 2 days, obvious cytopathic effects such as cell shrinkage, rounding, and aggregation appeared. As the number of passages increased, the cytopathic effects became increasingly obvious. After passage 130, it was named H18-R.

[0043] In order to obtain the full genome sequence of H18-R, the viral genome was divided into 12 fragments for amplification. The results of agarose gel electrophoresis showed that the 12 fragments had a single amplified band, and the fragment size was about 1.4kb, which was consistent with expectations. The sequences of the above fragments were spliced ​​to obtain the full genome sequence of H18-R, which was 14,486bp (excluding polyA). The full length of the H18-R genome was compared with the reference strains of each branch of PRRSV. The results showed that H18-R was a natural recombinant strain with lineage 1 strain as the main parent and lineage 8 as the secondary parent ( Figure 1 ).

[0044] During the domestication process, the H18-R genome had some amino acid deletions, located in the NSP2 coding region 1271-1795bp, and the nucleotide sequence of the deleted part is shown in SEQ ID NO.1. Bioinformatics software analysis showed that the antigenicity index of the deleted amino acid sequence (SEQ ID NO:2) was high. The deleted sequence can not only distinguish H18-R from the parent strain H18 and the wild strain, but also distinguish H18-R strain from other vaccine strains, and has differential diagnostic value.

[0045] Nucleotide sequence (SEQ ID NO: 1):

[0046] GGGACTCTGGCGTGCCGGGATGAGCTCCTCGATTTGTCTGCATCTTCGCAGACTGAAT

[0047] ACGAGGCTTCCCCTTTGGCGTTACCGCAGAGTGAGGACGCCCTGGCGATGAGGAGGC

[0048] AAGAAGCTGAGGAAACCCTGAGCGAAGCCTCGGGTGTGCCGGACGACATCAGATTGA

[0049] CACCCGTGTCGTCAAGCAGCTCCCTGTCAAGCGTAGAGATTACACGCCCAAAATACTC

[0050] AGCTCAAGCCATCATTGACACAGGTGGGCCCTGTTGTGGGCACCTCCAAGAGTTAAAG

[0051] GGGAAATACCTCGATGTGATGCGTGAGGGCTGTGATGCCACCAAGCTCGATGATCCTG

[0052] CCACGCAAGAATGGCTTTCTCGCATGTGGGACGGAGTGGACATGTTAACCTGGCGGA

[0053] GTACGTCCATTTTTCAAGCGCCTTTCATCTTGGCTGATAAGTTCAAATCTCTCCCGAAGATGATACTCGAAACACCACCGCCCTACCCTTGCGGGTTTGTGATG.

[0054] Example 2. Immune protection experiment of porcine reproductive and respiratory syndrome virus recombinant attenuated vaccine strain H18-R

[0055] 1. Materials and Methods

[0056] 1.1 Experimental animal grouping and vaccine immunization

[0057] Six 28-day-old piglets that were negative for PRRSV antigen and antibody and negative for classical swine fever virus (CSFV), pseudorabies virus (PRV) and porcine circovirustype 2 (PCV2) antigen were selected and randomly divided into a vaccine-immunized group and a non-immunized control group, with 3 pigs in each group, and the two groups were raised in isolation.

[0058] 1.2 Immunization and challenge experiments with recombinant attenuated vaccine strains

[0059] The pigs in the vaccine group were injected with PRRSV H18-R strain via the neck muscle, with the inoculation dose of 1 mL / head (10 5.0 TCID50 / mL). The non-immunized control group was injected with DMEM medium in the same manner and dose. On the 28th day after vaccination, all experimental pigs were injected with 3mL (10 5.0 TCID50 / mL).

[0060] 1.3 Body temperature measurement and clinical symptom observation of experimental pigs

[0061] After the experimental pigs were immunized with the recombinant attenuated vaccine, clinical manifestations were observed daily. After the pigs were inoculated with the H18 strong strain, clinical symptoms were observed and quantified daily, and body temperature was measured daily until 21 days after the virus challenge.

[0062] 1.4 Detection of PRRSV-specific antibodies in pig serum

[0063] The sera of all experimental pigs were collected on days 0, 7, 14 and 21 after immunization with the recombinant attenuated vaccine strain, and the PRRSV N protein level in the sera was detected using the IDEXX PRRSV antibody detection kit.

[0064] 1.5 Detection of serum viral load in experimental pigs

[0065] The sera of all experimental pigs were collected on the 7th, 10th, 14th and 21st days after inoculation with the strong H18 strain, and the viral load in the serum was quantified using the universal PRRSV-2 RT-qPCR method established in our laboratory.

[0066] 2. Results

[0067] The recombinant attenuated vaccine strain was used to conduct an immune protection experiment on 28-day-old piglets. 14 days after vaccination, the PRRSV N protein antibody in the pig serum turned positive (3 / 3), and the antibody level continued to increase thereafter ( Figure 2 ), indicating that the H18-R strain has good immunogenicity and can effectively activate the body to produce humoral immune response. Within 28 days after vaccination, the pigs in the vaccine immunization group showed no abnormal symptoms such as fever and respiratory symptoms, indicating that the H18-R strain has been domesticated in vitro and has no pathogenicity to pigs and has good safety. After all experimental pigs were challenged with the parental strong strain, clinical symptoms began to appear in the non-immunized control group pigs on the first day after the challenge, and the clinical symptoms disappeared on the 15th day after the challenge. The clinical symptoms were fever, cough, runny nose and other respiratory symptoms ( Figure 3 The body temperature of the pigs in the non-immunized control group increased significantly 2 to 11 days after the virus challenge, and all three experimental pigs had fever (body temperature>40.0℃) that lasted for 2 to 7 days. However, the pigs in the vaccine-immunized group did not show clinical manifestations such as fever and respiratory symptoms within 21 days after the virus challenge ( Figure 4 ), indicating that the H18-R strain can effectively resist the attack of the parental strong strain and effectively reduce the incidence of piglets. The serum was collected on the 7th, 10th, 14th and 21st day after the virus attack to detect the viral load. The viral load of the non-immunized control group was the highest on the 7th day after the virus attack, and then the viremia gradually decreased. The viremia of pigs in the H18-R immunized group remained at a low level 21 days after the virus attack, which was significantly lower than that of the non-immunized control group ( Figure 5 ), indicating that the H18-R strain can significantly reduce the viremia of pigs challenged with the virus and has a good immune protection effect.

[0068] Example 3. Establishment and application of ELISA method for differential diagnosis of attenuated vaccine strain H18-R strain

[0069] 1. Materials and Methods

[0070] 1.1 Plasmids and serum clinical samples

[0071] The sera of pigs immunized with vaccine strain H18-R, sera of pigs infected with parental strain H18, positive and negative sera of porcine reproductive and respiratory syndrome (PRRS), sera of specific pathogen-free (SPF) pigs, and positive sera of classical swine fever (CSF), porcine epidemic diarrhea (PED), pseudorabies (PR), porcine parvovirus infection (PPI) and porcine circovirus-2 (PCV2) were all stored in our laboratory; positive sera of African swine fever (ASF) were provided by the National African Swine Fever Professional Laboratory; clinical samples used in this experiment were all stored in our laboratory. pET-24a plasmid was stored in our laboratory.

[0072] 1.2 Analysis of the structure and antigenicity of the protein encoded by the deleted region

[0073] According to the comparison results of the vaccine strain H18-R sequence with the full-length sequence of PRRSV-2 NSP2 in GenBank (n=1242) and the unique deletion characteristics of its NSP2 region, the corresponding protein expression coding sequence was designed, and the Protean in DNASTAR software was used for analysis to predict its secondary structure and antigenic epitope. According to the indicators such as Antigenic Index and HydrophobicityPlot, the antigenicity as a protein antigen and the potential for soluble expression were preliminarily evaluated. In addition, the tertiary structure was predicted using Phyre2 software, and its spatial conformation was preliminarily predicted. The protein expression coding sequence designed according to the deletion region of H18-R strain NSP2 was optimized by Escherichia coli codons and synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd., and named D169 (SEQ ID NO: 2).

[0074] Amino acid sequence (SEQ ID NO: 2):

[0075] GTLACRDELLDLSASSQTEYEASPLALPQSEDALAMRRQEAEETLSEASGVPDDIRLTPVSS

[0076] SSSLSSVEITRPKYSAQAIIDTGGPCCGHLQELKGKYLDVMREACDATKLDDPATQEWLSR MWDGVDMLTWRSTSIFQAPFILADKFKSLPKMILETPPPYPCGFVM.

[0077] 1.3 Construction of pET-24a-D169 prokaryotic expression vector

[0078] After culturing the synthetic strain, the plasmid was extracted and double-digested with Nde I and Xho I at 37°C for 3 h together with the pET-24a plasmid. The target fragment and the vector fragment were connected, and the recombinant plasmid was named pET-24a-D169.

[0079] 1.3 Prokaryotic expression, purification and identification of target protein D169

[0080] The expression bacteria were induced to express in small quantities under different temperatures (16℃ and 37℃) and IPTG concentrations (0.1, 0.3, 0.5, 1.0mM), and the optimal expression conditions (37℃, 1mM IPTG) were explored. Then, large-scale induction expression was performed, and the D169 protein with high purity was obtained by nickel column affinity chromatography, and the protein concentration was determined by BCA method. Subsequently, mouse His-tagged mAb and PRRS positive serum were used as primary antibodies, and goat anti-mouse IgG (H+L) and goat anti-swine IgG (H+L) were used as secondary antibodies for Western Blot identification.

[0081] 1.4 Determine the optimal coating concentration of antigen and the optimal dilution of serum

[0082] The D169 protein concentration was determined by the BCA method, and the coating solution (carbonate buffer, adjusted to pH 9.6) was used for gradient dilution (20 μg / mL to 0.02 μg / mL), 100 μL per well, and coated overnight at 4°C. PRRS positive and negative sera were diluted 2-fold with serum diluent (1:25 to 1:400), 100 μL per well, and incubated at 37°C for 1 hour.

[0083] 1.5 Optimization of reaction conditions

[0084] The reaction conditions were optimized from the aspects of blocking solution type (5% skim milk, 1% and 2% BSA, 10% FBS), serum diluent type (PBS, 1% and 2% BSA, 10% FBS, 0.5% casein diluent), serum action time (30, 45, 60 and 120 min), enzyme-labeled antibody dilution (1:1000-1:16000, 2-fold dilution), enzyme-labeled antibody action time (30, 45, 60 and 120 min) and substrate development time (5, 10, 15 and 20 min). The reaction degree of PRRS positive and negative serum under different conditions was tested, the OD450nm value was measured and the P / N value was calculated, and the optimal reaction conditions were determined by comprehensive consideration.

[0085] 1.6ROC analysis to determine the critical value

[0086] 84 clinical animal test sera (IDEXX test kit test results show that 67 of them are PRRS positive sera and 17 are PRRS negative sera), according to the optimized D169-ELISA method, the OD450nm value is measured, and the S / P value of the serum sample under this method is calculated (OD450nm value of the sample to be tested-average OD450nm value of the negative control) / (average OD450nm value of the positive control-average OD450nm value of the negative control). According to the OD450nm value and the S / P value, the ROC (Receiver operating characteristic curve) curve analysis is performed with the aid of SPSS (Statistical product and service solutions) software, and according to the sensitivity and specificity of the D169-ELISA method, the positive and negative serum cut-off values ​​(Cut-off value) of the OD450nm value and the S / P value of the method after statistical processing are obtained respectively.

[0087] 1.7 Specificity, sensitivity and repeatability test

[0088] According to the established D169-ELISA method, the sera of pigs immunized with the vaccine strain H18-R and the sera of pigs infected with its parent strain H18, as well as the positive sera of PRRS, ASF, CSF, PR, PED, PPI and PCV2 were tested simultaneously, and a negative control (SPF pig serum) was set up to evaluate its specificity. The PRRS positive serum was diluted in a gradient of 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, and the sensitivity was evaluated by the method and the IDEXX kit. The purified D169 protein of the same batch and different batches was coated on the ELISA plate, and 10 PRRS positive sera were tested by this method. Each serum was tested 3 times, and the results were statistically analyzed. The coefficient of variation of the S / P value was calculated to evaluate its repeatability.

[0089] 1.8 Clinical serum compliance test and preliminary application

[0090] 175 clinical serum samples (139 samples of wild-type infected pig serum and 36 samples of other vaccine strains immunized pig serum) were tested simultaneously by D169-ELISA method and IDEXX kit, and the coincidence rate between the two was calculated based on the results. The parent strain HeB-108 infected pig serum (0, 7, 14, 21, 28, 35, 42, 49 dpi), commercial PRRS vaccine strains (HuN4-F112, GDr180, JXA1-R, PC, VR-2332 strains) immunized pig serum (0, 7, 14, 21, 28 dpi), clinical wild-type infected pig serum (8 samples) and vaccine strain H18-R immunized pig serum (0, 7, 14, 21, 28 dpi) were further tested, and the sensitivity and differential diagnosis potential of the method in clinical application were preliminarily analyzed according to the critical value judgment method.

[0091] 2. Results

[0092] By changing the temperature, IPTG concentration and other conditions, it was found that the overall expression level of the target protein D169 was the highest when cultured at 37°C, 1mM IPTG, and a shaking speed of 200r / min for 5h, and the expression level in the supernatant after ultrasonic disruption was the highest. SDS-PAGE identification results showed that a clear protein band appeared at about 24ku ( Figure 6 a). The target protein with high purity was obtained by nickel column affinity chromatography ( Figure 6 b), the protein concentration was determined by BCA method and was 0.045 g / L. The purified target protein was identified by Western Blot, using mouse His-tagged mAb as the primary antibody ( Figure 6 c) and using PRRS positive serum as primary antibody ( Figure 6d), the specificity was good, indicating that D169 was correctly expressed and had good reactogenicity.

[0093] D169 protein was diluted with coating solution, and PRRS positive serum and negative serum were diluted with serum diluent. The optimal coating concentration was determined to be 2.5 μg / mL according to the OD450nm value and P / N value, and the optimal serum dilution was 1:50 (Table 2). Through the exploration and optimization of each reaction condition, combined with the OD450nm value and P / N value of negative and positive serum, it was finally determined that the blocking solution was 5% skim milk, blocked at 37°C for 2h; the serum diluent was 0.5% casein diluent, and acted at room temperature for 45min; the enzyme-labeled antibody dilution was 1:1000, and acted at room temperature for 60min; the TMB colorimetric solution was acted at room temperature for 15min.

[0094] Table 2 Determination of optimal antigen coating concentration and optimal serum dilution

[0095]

[0096]

[0097] Note: P represents positive serum OD 450nm Value, N represents negative serum OD 450nm The underlined part is the response value corresponding to the optimal condition.

[0098] This method was used to test 84 clinical sera (the IDEXX test results showed that 67 of them were positive and 17 were negative). The S / P value was calculated based on the test results, and the ROC curve statistical analysis was performed. The results showed that the positive and negative critical value was 0.525, and the S / P value ≥ 0.525 was judged as positive, and the S / P value < 0.525 was judged as negative. Similarly, the positive and negative serum critical value corresponding to the OD450nm value was 0.159.

[0099] The established D169-ELISA method was used to detect the sera of pigs immunized with the vaccine strain H18-R and the sera of pigs infected with its parent strain H18, as well as PRRS-positive sera and other swine disease (ASF, CSF, PR, PED, PPI and PCV-2)-positive sera (SPF pig sera were used as negative controls). Only the sera of pigs infected with the parent strain and PRRS-positive sera were positive, while the others were negative ( Figure 7 a), i.e., the target protein D169 was used as the coating antigen, which neither cross-reacted with sera from other swine diseases nor with sera from swine immunized with the vaccine strain H18-R, indicating that this method has good specificity and can be used for differential diagnosis between vaccine strain H18-R immunization and other PRRSV infections.

[0100] The D169-ELISA method was used to detect 2-fold diluted PRRS positive serum. When the positive serum was diluted 1:800, the S / P value was 0.60, which was still positive ( Figure 7 b); At the same time, the results of the IDEXX test kit were used as a control, and it was found that the sensitivity of the two was close (the sensitivity of the IDEXX test kit was 1:1600), which further showed that the sensitivity of this method was strong. The D169 target protein prepared from the same batch and different batches was coated on the ELISA plate, and the D169-ELISA method was used to test 10 PRRS sera with different antibody levels three times. The test results were statistically analyzed using SPSS software, and it was found that the average intra-batch coefficient of variation was 3.13%, and all were less than 5%; the average inter-batch coefficient of variation was 6.90%, and all were less than 10% (Table 3), indicating that the method has good repeatability.

[0101] Table 3 Repeatability test of D169-ELISA method

[0102]

[0103]

[0104] The established D169-ELISA method and the PRRSV antibody detection kit of IDEXX were used to synchronously detect 175 clinical serum samples (139 serum samples of wild-type pigs infected with the virus strain and 36 serum samples of other vaccine strains immunized pigs). The test results are shown in Table 4. The positive rate detected by this method was 69.71% (122 / 175), the positive rate detected by the IDEXX test kit was 76.57% (134 / 175), and the positive coincidence rate of the two was 89.55% (120 / 134); the negative rate detected by this method was 30.29% (53 / 175), the negative rate detected by the IDEXX test kit was 23.43% (41 / 175), and the negative coincidence rate of the two was 95.12% (39 / 41); the overall coincidence rate was 90.86% (159 / 175). The above results show that the detection results of this method for the serum antibody levels of pigs infected with PRRSV other than the vaccine strain H18-R are close to those of the IDEXX kit, and it has high sensitivity and reliability in clinical application.

[0105] Table 4 Comparison of the test results of IDEXX kit and D169-ELISA method for serum samples

[0106] IDEXX Test Kits D169-ELISA method Compliance rate Number of negative samples 41 53(39*) 95.12％ Number of positive samples 134 122(120*) 89.55％ total 175 175(159*) 90.86％

[0107] Note: The numbers marked with "*" in brackets indicate the number of samples with consistent test results by the two methods.

[0108] The D169-ELISA method was used to test the sera of pigs infected with the parent strain with known clinical background (0, 7, 14, 21, 28, 35, 42, and 49 days after infection). The results showed that pigs began to test positive for D169 antibodies 21 days after infection, and D169 antibodies could still be detected 49 days after infection ( Figure 8 a). This method was used to detect the sera of pigs immunized with five commercial vaccines (HuN4-F112, GDr180, JXA1-R, PC, and VR-2332 strains) on 0, 7, 14, 21, and 28 dpi. The results showed that D169 antibodies could be detected in the sera of pigs immunized with these commercial vaccines after 21 days ( Figure 8 b). This method was used to test 8 samples of sera from wild-type pigs. The results showed that D169 antibodies were detected in all samples, and the OD450nm value and S / P value were both high ( Figure 8 c). In particular, the method was used to test the sera of pigs immunized with the H18-R strain (0, 7, 14, 21, and 28 dpi), and the results were all negative ( Figure 8 d). The above results further indicate that this method can detect antibodies against the protein D169 encoded by the deletion region in sera of wild strains and pigs infected with other vaccine strains except the vaccine strain H18-R, and can be used for clinical differential diagnosis.

Claims

1. An attenuated strain H18-R of arterivirus porcine reproductive and respiratory syndrome virus, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO.46203 and the deposit date October 17, 2024.

2. A porcine reproductive and respiratory syndrome virus vaccine, characterized in that: The vaccine comprises the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus according to claim 1.

3. The vaccine according to claim 2, characterized in that The vaccine also includes a pharmaceutical carrier or a pharmaceutically acceptable excipient.

4. Use of the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus according to claim 1 in the preparation of a vaccine or medicine for preventing porcine reproductive and respiratory syndrome.

5. An ELISA test kit for detecting porcine reproductive and respiratory syndrome virus and distinguishing the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus, characterized in that: The kit contains a protein with an amino acid sequence as shown in SEQ ID NO.

2.

6. The ELISA test kit according to claim 5, characterized in that The kit includes a first antibody and a second antibody, wherein the first antibody includes PRRS positive serum, and the second antibody is HRP-labeled goat anti-swine IgG (H+L).

7. Use of the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus as described in claim 1, the protein with an amino acid sequence as shown in SEQ ID NO.2, the microbial cells carrying the protein shown in SEQ ID NO.2, the coding gene SEQ ID NO.1 encoding the protein shown in SEQ ID NO.2, and the recombinant vector containing the coding gene SEQ ID NO.1 in the preparation of a diagnostic and detection kit for porcine reproductive and respiratory syndrome virus.

8. An antigenic peptide of reproductive and respiratory syndrome virus, characterized in that: The amino acid sequence of the antigen peptide is shown in SEQ ID NO.

2.

9. A nucleotide encoding the antigenic peptide according to claim 8.

10. Use of a recombinant vector or recombinant microbial cell containing the nucleotide according to claim 9 in the preparation of an ELISA detection kit for detecting porcine reproductive and respiratory syndrome virus and identifying and distinguishing the attenuated porcine reproductive and respiratory syndrome virus strain H18-R.

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