[Functional analysis of SNPs in porcine TLR4 gene]

Toll-like receptor 4 (TLR4) plays an important role in immune response and the polymorphism in it might affect protein signaling and host resistance/susceptibility to disease. This study was designed to characterize the functional relevance of 3 nonsynonymous single nucleotide polymorphisms (SNPs), c.611 T>A (p.Leu204His), c.1027 C>A (p.Gln343Lys), and c.1605 G>T (p.Leu535Phe), which were selected based on our previous studies. RT-PCR method was used to clone the complete coding sequence of porcine TLR4 gene and the PCR-based method was used to introduce the point mutation. The effects of 3 SNPs on the ligand recognition and signaling of porcine TLR4 were investigated in transiently transfected PK-15 cells using dual-luciferase reporter system and Western blotting method. At the same time, the distribution of c.1605 G>T among pig populations composed of Min pig, Yorkshire, Landrace, and Wild boar from northeastern China was studied by created restriction site PCR-RFLP method. The complete coding sequence of TLR4 gene in Min pig and 3 variants with single point mutations were obtained. Eukaryotic expression vectors containing different alleles of porcine TLR4 were constructed. SNP c.1605 G>T significantly decreased the TLR4 signaling (P<0.01) and the polymorphism only existed in Min pig and Wild boar from northeastern China with high frequencies. SNP c.1605 G>T in porcine TLR4 might affect the receptor function and host resistance/susceptibility to diseases.