| TLR4(Toll-like receptor 4)is an important pattern recognition receptors(PRRs),Which plays a leading role in mediating the transmembrane signal transduction pathway of lipopolysaccharide(LPS),it also participates in mediating the synthesis and release of a series of pro-inflammatory cytokines induced by LPS and triggers an excessive inflammatory response.In the G-pathogenesis mechanism has a crucial role,it has been confirmed that TLR4 is the most important pattern recognition receptor for LPS in humans and mice.As a transmembrane signal receptor,TLR4 can stimulate LPS signal Directly into the cell,it plays a key role that in mediate gram-negative bacteria and host reactions of LPS.To study the changes of TLR4 gene and intracellular inflammatory factors mediated by LPS is to clarify the basic approach of TLR4 gene in gram-negative bacterial infection.In this study,qianbei brown goat was selected as the experimental object.Primers were designed for the three exons of the goat TLR4 gene sequence in GeneBank.The three exons of SNPs in TLR4 were screened by PCR and direct sequencing;the TLR4 was cloned.The full-length CDS gene was constructed and the recombinant eukaryotic expression vector pEGFP-N1-TLR4 was constructed.The recombinant eukaryotic expression vector was transfected into mouse macrophages(Ana-1),and the GFP activity was detected 48 h after transfection and the transfected cells were stimulated with LPS,3,6,9,12 and 24 h after stimulation.The supernatant was collected and Elisa was used to detect intracellular inflammatory cytokines IL-1,IL-6 and TNF-? induced by LPS at different time intervals.The results of the study are as follows:(1)SNPs were screened for three exons of TLR4 gene in qianbei brown goat,and three SNPs were found: Intron1 A+212C,Intron1 G+219A,and Exon3 C+8209G.The C+8209G site causes the encoded amino acid to be mutated from leucine to proline.(2)The full-length CDS of TLR4 gene was cloned successfully from qianbei brown goat and the recombinant expression vector pEGFP-N1-TLR4 was constructed.(3)The relative expressions of TLR4 and TLR2 genes in macrophages of transfected andnormal mice were detected by qRT-PCR.The results showed that TLR4 mRNA expression was significantly increased in positive transfection group compared with TLR2(P<0.05).The experiments were performed on mouse macrophages 48 h after transfection which induced by LPS with 0 ng,100 ng,200 ng,500 ng,1000 ng,and 2000 ng,respectively,3 h,6 h,9 h,and 12 h after induction.The cell supernatant was collected at h and 24 h.Elisa detection was performed on the three inflammatory factors IL-1,IL-6 and TNF-? in the supernatant.The results showed that:(1)IL-1,IL-6 and TNF-? of secretion were different in different LPS concentrations.When LPS concentrations were 100 ng and 200 ng,IL-1 secretion was higher than that of the control group.The difference was significant(P<0.05).When the concentration of LPS was greater than500 ng,the secretion of IL-1 was significantly different from that of the control group(P<0.01),And the secretion of IL-1 and LPS showed a dose-dependent manner.(2)The secretion of IL-6 increased with time.Under the stimulation of 100 ng LPS,there was no significant difference between the secretion of IL-6 and the secretion of IL-6 in the control group(P>0.05).When the concentration of LPS was greater than 100 ng The secretion of IL-6was significantly different from that of the control group(P<0.05),and the secretion of IL-6 was still increasing.(3)The secretion of TNF-? at 100 ng of LPS reached a maximum at 12 h,and the secretion of TNF-? decreased at 24 h.When LPS concentration was 100 ng,there was a significant difference between TNF-? secretion and TNF-? secretion in the control group(P<0.01),indicating that the intracellular inflammatory intensity was highest,that is,the TLR4 gene was maximally involved in LPS of response.With the increase of LPS concentration,the secretion of TNF-? also decreased,compared with the control group,the difference was not significant(P>0.05). |
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