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| Expression of Goat (Capra hircus) TLR4 Gene in Mouse (Mus musculus) Macrophages and Its Correlation with the Expression of TLR2 Gene |
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Abstract The Toll like receptor family (TLRs) is an important type of pattern recognition receptor (PRRs), which has been widely studied in recent years. TLRs activates cascade reaction in the process of signaling pathway by identifying the invasion of pathogenic microorganisms, inducing tissue or organism to produce cytokines and inflammatory factors, which plays an extremely important role in the host against pathogen infection. The purpose of this study was to explore the correlation between the overexpression of goat (Capra hircus) TLR4 gene in mouse macrophages and the expression of TLR2 gene. In this experiment, the whole length of CDS of goat TLR4 gene was cloned by reverse transcription PCR (RT-PCR) amplification combined with T cloning method. The pEGFP-N1-TLR4 eukaryotic expression vector was transiently transfected to mouse macrophages (Ana-1) with lipofectamine3000TM after identification the eukaryotic expression vector by connection, transformation and double enzyme digestion. The total RNA was extracted after transfection, and TLR4 and TLR2 gene mRNA in transfected cell Ana-1 were detected by qRT-PCR and the differential expression was analyzed by the software of SPSS 18.0. Results showed that the TLR4 gene CDS region was 2 537 bp, 3 mutation sites (T426C, C1254T, G1515A) in the CDS region of TLR4 gene were found, and 3 mutations did not cause changes in amino acids, which belonged to synonymous mutations. The eukaryotic expression vector of pEGFP-N1-TLR4 were successfully transfected and expressed in the mouse macrophages, the qRT-PCR test showed that the expression of TLR4 and TLR2 was positive group＞negative group＞control group (blank), in control group, there was no significant difference in mRNA expression between TLR4 and TLR2 gene (P＞0.05), while mRNA expression in TLR4 positive group was significantly higher than that in TLR2 positive group (0.01＜P＜0.05). The expression level of TLR2 gene mRNA in the positive group was also significantly higher than that in the blank group (0.01＜P＜0.05). The results showed that the overexpression of TLR4 could promote the TLR2 gene. This study provide a basis for further study of the interaction between TLR4 and TLR2 genes and their intracellular expression mechanism.
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Received: 02 April 2018
Published: 24 October 2018
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