SNP molecular marker of prrx2 gene associated with antibody level of african swine fever virus, primer and application

Abstract

This invention belongs to the field of molecular marker technology, specifically relating to SNP molecular markers, primers, and applications of the PRRX2 gene associated with African swine fever virus (ASFV) antibody levels. The SNP molecular marker is located at 143 bp of exon 3 of the porcine PRRX2 gene (genotype XM_005654574.2), exhibiting T / C polymorphism. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3. The molecular marker provided by this invention is closely associated with ASFV antibody levels in pigs. Compared to pigs with the TC / CC genotype at this locus, pigs with the TT genotype have higher ASFV antibody levels in their serum. This molecular marker can be applied to breed high-quality breeding pigs resistant to ASFV, thereby effectively improving the economic benefits of pig farming.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to SNP molecular markers, primers, and applications of the PRRX2 gene associated with African swine fever virus antibody levels. Background Technology

[0002] African swine fever (ASF) is a highly contagious disease of pigs caused by the African swine fever virus (ASFV). Clinical features include high fever, lethargy, anorexia, cyanosis of the skin, and hemorrhage in various organs. This disease poses a serious threat to the pig farming industry.

[0003] Currently, vaccine development for ASFV still faces certain challenges. The immunogenicity of inactivated vaccines, subunit vaccines, and gene-deleted vaccines has not been fully elucidated, and they do not provide complete protection after vaccination. Studies show that key structural proteins of ASFV, such as the p72 protein located in the viral capsid and the CD2v protein located in the viral envelope, are important antigenic proteins that elicit humoral immune responses. The expression of the p72 gene occurs in the late stage of viral infection and is regulated by the late promoter of the virus. P72 antibodies can be detected as early as day 17 after African swine fever virus infection, and their long duration and high antibody levels are markers of persistent viral infection or the formation of immunity.

[0004] After vaccination against African swine fever (ASF), the levels of ASF virus antibodies produced in pig herds vary considerably. Approximately 67% of individuals have high levels that provide complete protection, while the remainder have low levels that do not provide complete protection. Therefore, it is necessary to identify SNP molecular markers of antibody formation-related pathway genes and apply them to the breeding of pigs with high ASF virus antibody levels. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides SNP molecular markers, primers, and applications of the PRRX2 gene associated with African swine fever virus antibody levels, aiming to provide new technical means for the genetic improvement of pig breeds.

[0006] This invention first provides a molecular marker for the PRRX2 gene associated with African swine fever virus antibody levels and its application in pig breeding; secondly, it provides a primer set for amplifying the molecular marker for the PRRX2 gene associated with African swine fever virus antibody levels, the application of the primer set, and specific application methods.

[0007] The first aspect of the present invention provides an SNP molecular marker of the PRRX2 gene associated with African swine fever virus antibody levels. The SNP molecular marker is located at 143 bp of the third exon of the porcine PRRX2 gene with gene version number XM_005654574.2 and has T / C polymorphism. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3.

[0008] .

[0009] A second aspect of the present invention provides a primer set for amplifying the SNP molecular marker associated with African swine fever virus antibody levels as described in claim 1, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2.

[0010] SEQ ID NO. 1: TGTCCCGTGACTTTGTGCAT.

[0011] SEQ ID NO. 2: CTGGCTGCTCTCCAACACC.

[0012] A third aspect of the present invention provides a kit for identifying SNP molecular marker genotypes associated with African swine fever virus antibody levels, the kit comprising the primer set described in the second aspect of the present invention.

[0013] Furthermore, the kit also includes DNA extraction reagents, DNA purification reagents, PCR reaction reagents, agarose solution, electrophoresis buffer, and staining solution.

[0014] The fourth aspect of this invention provides the application of the SNP molecular marker associated with African swine fever virus antibody levels, the primer set, or the kit in pig breeding.

[0015] Furthermore, the application includes the following steps:

[0016] The primer set was used to amplify the pig DNA template by PCR to obtain the PCR product.

[0017] The PCR products were subjected to agarose gel electrophoresis and sequencing.

[0018] The African swine fever virus antibody levels in pig breeds with the TT genotype (T / C polymorphism at 143 bp of exon 3 of the porcine PRRX2 gene) were higher than those in pig breeds with the TC or CC genotypes.

[0019] Furthermore, the reaction system for PCR amplification is as follows:

[0020] 10 μL of 2×Taq Master Mix, 1 μL of DNA template, 0.3 μL of upstream primer, 0.3 μL of downstream primer, and 8.4 μL of ddH2O.

[0021] Furthermore, the reaction procedure for PCR amplification is as follows:

[0022] Pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 25 s, annealing at 61℃ for 18 s, extension at 72℃ for 15 s, cycled 39 times; extension at 72℃ for 5 min, stored at 10℃.

[0023] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0024] This invention provides a SNP molecular marker associated with African swine fever virus (ASFV) antibody levels. The SNP marker is located at 143 bp of exon 3 of the porcine PRRX2 gene (version XM_005654574.2) and exhibits a T / C polymorphism. The nucleotide sequence of the SNP marker is shown in SEQ ID NO.3. This invention, through in-depth research on key genes in the porcine antigen presentation pathway, provides for the first time a novel site significantly associated with ASFV antibody levels. Validation revealed that the T / C mutation at 143 bp of exon 3 of the PRRX2 gene (XM_005654574.2) is negatively correlated with serum ASFV antibody levels in pigs.

[0025] The molecular markers provided by this invention can be applied to the breeding of pigs resistant to African swine fever, thereby improving breeding efficiency, increasing disease resistance, improving animal health, and reducing unnecessary feeding and time costs. Precision breeding targeting molecular markers can reduce culling rates caused by undesirable traits, further saving production costs. Furthermore, this invention has significant scientific value for studying the molecular mechanisms affecting African swine fever virus antibody levels. Attached Figure Description

[0026] Figure 1 This is a correlation analysis image of the expression level of PRRX2 target genes in lymph nodes and the PRRX2 binding sites of the target genes. The green dots in the figure represent a two-dimensional scatter plot of the binding site scores of PRRX2-regulated target genes and the expression levels of the target genes. The size of the green dots represents the weighting value. The yellow curve in the figure is a weighted fitting curve of the transcription factor binding site scores of target genes and the expression levels of target genes. The green curve is the average expression level of target genes.

[0027] Figure 2 The sequencing results are shown for three pigs with different PRRX2 genotypes; the T143C mutation site is indicated by the blue box.

[0028] Figure 3 The graph shows the effect of the T143C mutation site in the PRRX2 gene on the antibody level trait of African swine fever virus; the vertical axis represents the antibody level of African swine fever virus (S / N), and the horizontal axis represents the genotype. Detailed Implementation

[0029] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0030] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0031] The antigen presentation signaling pathway is closely related to the production of antibodies against African swine fever (ASF), involving the activation of antigen-presenting cells, B cells, and helper T cells. ASF virus antibody levels are an important marker of an animal's resistance to ASF and its overall health. However, the relatively limited number of molecular markers currently developed that directly correlate with ASF virus antibody levels restricts the application of marker-assisted breeding techniques in the development of ASF-resistant pig breeds.

[0032] This invention first analyzed transcriptome sequencing data from pigs infected with African swine fever virus (ASFV) using GRIT / FLAVER software. The analysis revealed that PRRX2 is a key transcription factor regulating ASFV infection. Among the target genes regulated by PRRX2, ANAPC5, UBE2K, KIF11, and DCTN6 are associated with antigen presentation and antibody production signaling pathways. Comparison of resequencing data from 30 Duroc × Landrace × Large White pigs revealed a T / C polymorphism at 143 bp of exon 3 of the PRRX2 gene (XM_005654574.2). Primers designed for this specific region were used to genotype DNA samples from 206 pigs, revealing that the genotype frequencies of the PRRX2 gene SNP sites showed a trend of TT>TC>CC. Subsequently, it was verified that the SNP molecular markers provided in this invention are negatively correlated with the level of ASFV antibodies in pig serum and are easily detectable.

[0033] Example 1: Obtaining the PRRX2 gene fragment and SNP molecular markers

[0034] Transcriptome sequencing analysis was performed on six representative pigs, including three African swine fever virus-infected pigs and three uninfected control pigs. The expression levels of differentially expressed genes in the infected / control groups were calculated, and the weighted Kendall correlation test using GRIT / FLAVER software was used to examine the correlation between the expression levels of differentially expressed genes in pigs and the transcription factor binding sites in each gene sequence.

[0035] The results are as follows Figure 1 As shown, the higher the score of the transcription factor binding site (gene-set) in the gene sequence, the higher the expression level (gene-list) of the gene. For genes containing the PRRX2 binding site, there is a significant positive correlation between their expression level and the transcription factor binding site (p<0.05).

[0036] Comparison of resequencing data from 30 Duroc × Landrace × Large White pigs revealed a T / C polymorphism at 143 bp of exon 3 of the PRRX2 gene. Sequencing was performed on three individuals with different genotypes using the Sanger method, and the comparison results are as follows. Figure 2 As shown in the image, the yellow box indicates the T143C mutation site.

[0037] Blood samples from 206 pigs infected with African swine fever virus were collected from farms in Jingzhou City, Hubei Province. Serum and cells were separated by centrifugation, and genomic DNA was extracted from the cells using a peripheral blood DNA extraction kit purchased from Beijing Solarbio Co., Ltd.

[0038] Based on the nucleotide sequence of the PRRX2 gene with version number XM_005654574.2 published in the GenBank database, specific primers for the SNP region of the PRRX2 gene were designed using Oligo software, and PCR amplification was performed using genomic DNA from 206 blood cells as templates.

[0039] The upstream primer sequence for the PRRX2 gene is shown in SEQ ID NO.1; the downstream primer sequence is shown in SEQ ID NO.2.

[0040] SEQ ID NO. 1: TGTCCCGTGACTTTGTGCAT.

[0041] SEQ ID NO. 2: CTGGCTGCTCTCCAACACC.

[0042] Table 1 shows the specific parameters of the PCR reaction system.

[0043] Table 1 PCR reaction system

[0044] Element Content (μL) 2×TaqMasterMix 10 Upstream primer (10 μM) 0.3 Downstream primer (10 μM) 0.3 DNA template 1.0 <![CDATA[ddH2O]]> 8.4

[0045] The PCR amplification procedure is shown in Table 2.

[0046] Table 2 PCR amplification program

[0047]

[0048] Note: "-" indicates that this item is not available.

[0049] Four μL of each PCR product was taken for agarose gel electrophoresis. After imaging, the PCR products containing a single band were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing results were compared and analyzed using SeqMan software to identify mutation sites in the sequences, and the mutation type of T143C at the corresponding sites in each individual was recorded.

[0050] The T143C mutation site of the PRRX2 gene showed three genotypes in 206 samples tested, with genotype frequencies of TT: 0.78; TC: 0.21; CC: 0.01, showing a trend of TT>TC>CC. The frequency of allele T was 0.88, and the frequency of allele C was 0.12, with the T allele frequency being greater than the C allele frequency.

[0051] Example 2: Validation of SNP molecular markers associated with African swine fever virus antibody levels

[0052] To establish the relationship between the PRRX2 gene and the level of African swine fever virus (ASFV) antibody, the level of ASFV p72 antibody in 206 swine serum samples from Example 1 was determined using a blocking ELISA method. The results were expressed as the S / N value (sample OD). 450nm Value / Negative Control OD450nm The average value (S / N) indicates that a higher S / N value corresponds to a lower antibody level, and vice versa. Results showed that 150 swine serum samples had high antibody levels (S / N ≤ 0.5, positive), 6 swine serum samples had low antibody levels (S / N ≥ 0.6, negative), and 50 swine serum samples had S / N values ​​between 0.5 and 0.6.

[0053] The association analysis of SNP sites in the PRRX2 gene and African swine fever virus antibody levels in each individual was performed using the ANOVA program in R 4.1 software. The results are as follows: Figure 3 As shown in the figure, the S / N value of African swine fever virus antibodies in 160 serum samples with the TT genotype was 0.46±0.05, the S / N value in 43 serum samples with the TC genotype was 0.51±0.06, and the S / N value in 3 serum samples with the CC genotype was 0.73±0.15. The serum African swine fever virus antibody levels showed a significant difference between TT and TC, and then between TC and CC (p<0.05). This is basically consistent with the results of the blocking ELISA method, proving that the SNP molecular markers screened in Example 1 of this invention can be used to detect African swine fever virus antibody levels.

[0054] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0055] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a primer set of SNP molecular markers associated with African swine fever virus antibody levels in the preparation of reagents for identifying African swine fever antibody levels, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO. 3, wherein the base at 353bp has a T / C polymorphism; the T / C polymorphism of the SNP molecular marker at 353bp in SEQ ID NO. 3 indicates that the African swine fever virus antibody level in pig breeds with the TT genotype is higher than that in pig breeds with the TC or CC genotypes. The nucleotide sequence of the upstream primer of the primer set is shown in SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2; The antibody level refers to the antibody level against the p72 protein.

2. The application of the primer set according to claim 1 in the breeding of pigs resistant to African swine fever virus.

3. The application according to claim 2, characterized in that, Includes the following steps: The primer set described in claim 1 was used to perform PCR amplification on a pig DNA template to obtain PCR products; The PCR products were subjected to agarose gel electrophoresis and sequencing. The detection showed that the African swine fever virus antibody level in pig breeds with the T / C polymorphism at 353bp of SEQ ID NO. 3, as described in claim 1, was higher than that in pig breeds with the TC or CC genotypes.

4. The application according to claim 3, characterized in that, The PCR amplification reaction system was as follows: 10 μL of 2×Taq MasterMix, 1.0 μL of DNA template, 0.3 μL of upstream primer, 0.3 μL of downstream primer, and 8.4 μL of ddH2O.

5. The application according to claim 3, characterized in that, The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 25 s, 61℃ annealing for 18 s, 72℃ extension for 15 s, for 39 cycles; 72℃ extension for 5 min, and storage at 10℃.

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