The Mutation Of TLR4 Gene In Pig And Function Analysis

The Toll-like receptors (TLRs) family encodes single-spanning membrane proteins having an extracellular domain chiefly composed of leucine-rich repeats(LRRs), As one member of TLRs,Toll-like receptor 4(TLR4) gene was discovered in the late nineties,which is 2526bp in CDs,contains three exons. TLR4 is required for detection of Gram negative bacterial infections by binding lipopolysaccharide (LPS) and for the initiation of inflammatory signaling. LPS is a major component of the outer wall of Gram negative bacteria, serving as the key ligand for immune cell recognition and activation in response to infection. If be invaded by LPS,binding of the ligand to the extracellular domain of TLR4 initiates a complex signal-transduction cascade of host genes includeing nuclear factor-κB and pro-inflammatory cytokines.This study was designed to detect the polymorphisms by PCR-SSCP in six breeds of pigs; construct the expression vector of wild type and mutation in 611 bp,then were transfect to the epithetlial cells of pig kidneys(PK-15) and induced by LPS; the result were detected with the constructed dual-luciferase vectors and Real-time PCR.The main results are as follows:1 5 SNPs in exon 3 and 3'UTR of TLR4 gene were found by PCR-SSCP method,and there were 3 missenses.Population genetics analyses showed that in each polymorphic locus,there was extremely significant difference among 6 breeds in the distribution of genotypes(P<0.01).2 The TLR4 gene CDs 611bp were changed from T to A by in vitro site-directed mutagenesis,and the wild type and mutation expression vectors were cloned.3 The plasmid was mixed with pNF-κB and pHRL plasmid in 10:10:1,then were transfected to PK-15 cell 24h later induced in LPS for 12h, NF-κB transcript activation showed higher(P<0.05).4 The results showed that transfection of PK-15 cells with wild-type TLR4 resulted in strong NF-kB activation compared with mutation type (P<0.05),and had extremely significant different with LPS-induced (P<0.01). Wild-type TLR4 resulted in strong LPS-induced NF-kB activation. The mutated TLR4 had no significant different in reduced activation of NF-kB activation.5 The expression of IL-6 gene was analyzed by Real-time PCR. The results showed that transfection of PK-15 cells with wild-type TLR4 resulted in strong IL-6 expression level compared with mutation type (P<0.05), and had extremely significant different with LPS-induced (P<0.01). Wild-type TLR4 could give raise up the expression of IL-6 in strong LPS-induced. The mutated TLR4 had no significant different in reduced the expression of IL-6 gene.