Abstract: In this study, IRF3 gene was chosen as a candidate gene for evaluating its effect on porcine immune traits. A SNP (HQ026024: g.4252T>C) in exon7 of IRF3 gene was demonstrated by PCR-SSCP analysis and sequencing. Immune traits including IFN-γ and IL10 concentrations in serum were measured when the pigs are at 20th day of age. The further association analysis of SNP genotypes with immune traits were also conducted in 3 pig breeds including Large white, Landraces and Chinese indigenous breed Dingyuan black pig. The results indicated that the SNP of IRF3 gene had highly significant effects on level of IFN-γ and IL10 (day 20) in serum (p = 0.0344; p = 0.0121) and ratio of IFN-γ to IL10 (day 20) in serum (p = 0.0315). The results suggested that the IRF3 gene could be regarded as a molecular marker gene for genetic selection of these immune traits in the pig breeding program.

INTRODUCTION

Interferon Regulatory Factors (IRFs) are a family of transcription involved in interferon-inducible genes regulation, viral response, cytokine signaling, cell growth regulation and hematopoietic development (Nguyen et al., 1997; Taniguchi et al., 1995). The IRF gene family consists of nine members encoding transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain (Taniguchi et al., 2001). Most IRFs regulate the expression of IFN-α and -β after viral infection. IRFs play important roles in viral immune response, cytokine signaling, cell growth regulation and hematopoietic development (Nguyen et al., 1997; Honda and Taniguchi, 2006) thus, IRFs could be important marker genes for porcine immune response and disease susceptibility due to their drivers and potent effects on the immune system.

Cytokines are important mediators of the immune responses and vary with health and disease status. IFN-γ is critical for innate and adaptive immunity against viral and intracellular bacterial infections and for tumor control (Schoenborn and Wilson, 2007). IL-10 has pleiotropic effects in immunoregulation and inflammation. It down-regulates the expression of Th1 cytokines, MHC class II antigens (Redpath et al., 1999; Lohoff and Mak, 2005). Differences in levels of these immune traits and their ratios in serum among individuals under same conditions provide evidences for genetic control. Identification of these genes and their potential functional mutation on these immune variants may help to improve immune capacity in pigs. Interferon Regulatory Factor 3 (IRF3) gene is one of nine members of IRF-family and pays an important role in IFN regulation. Considering the effects on IFN immune response and cytokines signaling pathway of IRF3 gene, we firstly investigated polymorphisms of all exons region of porcine IRF3 gene and then analyzed the association between the SNP and immune traits (IFN-γ, IL-10, IFN-γ/IL-10) in serum to evaluate the possible effect of the IRF3 gene in 3 pig populations including Large white, Landrace and Chinese indigenous breed Dingyuan black pig.

MATERIALS AND METHODS

Animals and sampling: The animals consisted of 258 pig lets distributed in 3 pig breeds including big white pig (96), Landrace (89), Dingyuan black pig (73). Big white pigs and Landrace were from the experimental farm of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. Dingyuan black pigs were from breeding stock farm in Dingyuan county, Anhui province, China. The blood samples were collected from each piglet 1 day before the vaccination (day 20). All blood samples were directly injected into VACUETTE^® serum clot activator tubes. Ear tissue samples of all pigs were also collected for DNA extracting.

Measurement of IFN-γ and IL-10 concentration in serum: Immune traits including IFN-γ and IL-10 concentrations in each serum sample were measured using a commercial ELISA kit (Biosource, Carlsbad, California) according to the manufacturer’s instructions. All samples were arranged randomly in each plate and a standard curve was fitted for each plate to calculate IFN-γ and IL-10 concentrations in each serum sample.

Polymorphism detection and PCR-SSCP: Genomic DNA was isolated from the ear tissue sample using phenol/chloroform extraction and ethanol precipitation (Sambrook et al., 1989). Base on the cloned porcine IRF3 genomic DNA sequence (GenBank Accession No.: HQ026024), seven specific primers which spanned seven exons region of the IRF3 gene were designed by software Oligo6.0 (Table 1). The PCR was carried out in a total volume of 25 μL including 50-80 ng of template DNA, 10 pmol of each primer, 250 μM dNTPs, 2.5 μL 10xPCR buffer (with MgCl₂) and 1.5 U Taq polymerase (TaKaRa Biotechnology, China). PCR was performed in a MJ research PTC-200 Thermal Cycler (BIO-RAD, USA) under the following reaction procedure: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at X°C for 45 sec (X was shown in Table 1) and extension at 72°C for 1 min with a final extension step at 72°C for 5 min. PCR-SSCP method was used to find the mutation site in the amplified regions. Aliquots of 6 μL PCR products were mixed with 6 μL denaturing solution (95% formamide, 25 mM EDTA, 0.025% xylene-cyanole and 0.025% bromophenol blue), heated at 98°C for 10 min and then chilled in ice immediately. Denatured DNA was subjected to 12% Polyacrylamide Gel Electrophoresis (PAGE) in 1xTBE buffer and constant Voltage (130 V) for 15 h at a constant temperature of 4°C. After the process above, the gels were stained with 0.1% silver nitrate and visualized with 2% NaOH solution (containing 0.1% formaldehyde). The PCR products which represented different PCR-SSCP genotypes including both homozygous and heterozygous genotypes were purified with the Gel Midi PCR DNA purification kit (Tiangen Biotechnology, China) and sequenced using the ABI377 sequencer. Those tested sequences were aligned by using the DNAMAN software (Version 5.2.2) to identify the mutation site.

Table 1:	Specific primers used for SNP detection of the porcine IRF3 gene

Association analysis: Association analysis between genotypes of the SNP and immune traits were tested by using SAS9.1.3 software based on the general linear model. The general linear model is as follows:

Where:

RESULTS AND DISCUSSION

SNP identification and genetic variation analysis: Sequence comparisons among 3 pig breeds revealed one SNP (HQ026024: g.4252T>C) in exon 7 of the porcine IRF3 gene and the three genotypes of the SNP were also clearly obtained (Fig. 1). The SNP (HQ026024: g.4252T>C) of IRF3 is not missense mutation and not to induce amino acid substitution. A total of 258 DNA samples from animal populations were genotyped and allele frequencies were determined for each breed (Table 2). Genetic variation analysis demonstrated that allele frequencies were not significantly different among 3 pig breeds.

Fig. 1:	The PCR-SSCP electrophoresis patterns for the SNP (HQ026024: g.4252T>C) of porcine IRF3 gene. Lane TT, TC and CC represent different genotypes

Table 2:	Genotype frequencies and allelic frequencies of the IRF3 gene determined by PCR-SSCP in 3 pig populations

Table 3:	Association analysis of the SNP genotypes of IRF3 gene with immune traits in 3 pig populations
a. bSigned by small letters differ significantly at p<0.05; A, Bmeans signed by capital letters differ significantly at p<0.01

The allele T is obviously dominant in 3 detected breeds. In contrast, the allele C has lower frequencies and CC genotype is not detected in Chinese indigenous breed Dingyuan black pig.

Association analysis of immune traits and SNP genotypes: We performed an association study to determine whether the SNP affected immune traits in 3 pig populations. Association analysis results showed the SNP of IRF3 gene had significant effects on level of IFN-γ and IL10 (day 20) in serum (p = 0.0344; p = 0.0121) and ratio of IFN-γ to IL10 (day 20) in serum (p = 0.0315) (Table 3). Further, analysis results also showed that IFN-γ and IL10 level in serum of pigs with TT genotype were significantly higher than those of pigs with CC genotype (p<0.05) but ratio of IFN-γ to IL10 with CC genotype were significantly higher than those of pigs with TT genotype (p<0.05). The ratio of IFN-γ/IL-10 production reflects the capacity to activate or inhibit monocytic and T lymphocytic functions and a higher ratio has also been shown to be associated with depressive disorders (Maes, 1999).

The lower ratio of IFN-γ to IL-10 of Dingyuan black pig should be explained for Chinese indigenous breed may have better T lymphocytic balance and immune capacity than western commercial pig breeds. The results also implied that genetic background is one of the most important influences of immune traits. Interferon Regulatory Factor 3 (IRF3) plays an important role in initiating cellular interferon-stimulated gene-mediated antiviral responses. IRF 3 contributes to the host response during pseudomonas aeruginosa lung infection in mice (Carrigan et al., 2010).

In human, IRF3-dependent pathways are critical for control of herpes simplex virus type 1 central nervous system infection (Menachery et al., 2010); IRF3 gene are also revealed negative regulation of intracellular hepatitis C virus replication (Yamashiro et al., 2006) and polymorphisms of IRF3 gene are also association with lung cancer risk, systemic lupus erythematosus (Akahoshi et al., 2008; Shen et al., 2009).

In pig, another INF regulatory factor (IRF1) has been reported significantly associated with cytokine traits (Liu et al., 2009) and Classical swine fever virus Npro interacts with IRF3 gene and induces its proteasomal degradation (Bauhofer et al., 2007). While the SNP (HQ026024: g.4252T>C) of IRF3 in this study is not missense mutation or not to induce amino acid substitution but synonymous SNPs can affect protein expression by alteration or increase in the stability of the mRNA (Capon et al., 2004) and a silent polymorphism changes substrate specificity (Kimchi-Sarfaty et al., 2007).

The immune system plays an essential role in disease resistance of animals. The genes affect the response of immune system could be researched as the candidate genes. The results indicated that the SNP of IRF3 may be regarded as a genetic marker gene for immune traits with effects on IFN-γ, IL-10 and ratio of IFN-γ to IL-10 in serum. However, the number of pigs analyzed is limited further investigation is also required among other populations of pigs to confirm the association between the SNP and immune traits.

CONCLUSION

In this study, a SNP in exon 7 of the IRF3 gene was identified by PCR-SSCP method. Further association analysis between the SNP and immune traits was also performed, the results showed that the SNP of IRF3 gene was significantly associated with IFN-γ to IL-10 level of serum in 258 pigs from 3 pig populations. All these results will provide a foundation for further studies of the porcine IRF3 gene and may as a candidate gene for marker-assist selection in pig disease resistance breeding program.

ACKNOWLEDGEMENT

This study was supported by Scientific Technology Plan of Education Department of Anhui province (Grand No. 2006KJ034C).