Association of the porcine C3 gene with hemolytic complement activity in the pig

Complement component C3 is the major key molecule of the complement system which is involved in phagocytosis, inflammation and immunoregulation to destroy infectious microorganism. The aim of this study was to identify the cDNA sequence of porcine C3 and 5' and 3' flanking regions and to screen for polymorphism that enable to carry out association analysis between C3 and complement activity and other immune response traits. The porcine C3 cDNA sequence (AccNo: AF154933) was obtained from liver mRNA. The 5132 bp cDNA contains an open reading frame coding for 1661 amino acids and has high homology to human C3. The 487 bp of 5' flanking region of porcine C3 was isolated from a genomic DNA library. Regulatory elements, such as TATA-box, IL-1- and IL-6-binding sites similar to that of human C3 promoter were identified. The polymorphism study of porcine C3 revealed 6 polymorphic sites, including 5 SNPs and one simple sequence length polymorphism (SSLP). Two SNPs were found in the 5' flanking region and genotyped by PCR-SSCP. Three SNPs located in the coding region were genotyped by PCR-PFLP using restriction enzyme Taq I and Hsp92 I and by allele specific PCR. The SSLP located 70 bp downstream of the stop codon within the 881 bp of 3' flanking region that were obtained was genotyped by PAGE. The polymorphisms of porcine C3 gene were found to segregate within the DUMI F2 resource population that is based on reciprocal cross of Duroc and Berlin Miniature Pig. The allelic variations were also found in Thai native pigs whereas no polymorphisms could be detected in 86 commercial pig breeds (Duroc, German Landrace, Large White, Pietrain). Association between the porcine C3 and complement activity was analysed in 417 animals of the DUMI F2 resource population, which were immunized with Mycoplasma, Aujeszky and PRRS vaccines. Hemolytic complement activity was found to increase from 49.5 before immunization to 63.1 U/ml after immunization in the alternative pathway and from 39.2 to 59.6 U/ml in classical pathway. C3 was found to be significantly associated (p<0.05) with both alternative and classical pathway complement activities (ACP, CCP). Animals with the more frequent haplotype: present in Duroc and other commercial breeds exhibit higher ACP and CCP (+5.8 U/ml and +6.9 U/ml) than the animals with haplotype that was specific to some Berlin Miniature Pigs. The association of C3 with complement activity - a defence mechanism active against many pathogenic agents - promote the importance of C3 as candidate gene for natural resistance to microorganisms. (Texte intégral)