| The present study was conducted to explore the influence of heat stress on the transcription level of TLR2 mRNA, and TLR4 mRNA as well as the concentration of serum cortisol, IL-2 and IL-8 in PBMC of Bama-miniature pigs. Whilst, the changing of TLR2 mRNA, TLR4 mRNA ,TLR4-ASV, IL-12 and IFN-γin PBMC that were cultured in vitro after heat shock were also detected through quantitative real-time PCR, ELISA, radioimmunoassay and cell culture. The results showed that:(1)Toll-like receptor 2 gene cDNA consists of 2358 base pairs, coding 785 amino acids.The molecular weight and the isoelectric point of TLR2 protein is 89.48 kD and 7.52, respectively, and a signal peptide.15 bases of Toll-like receptor 2 gene in Bama-miniature pigs mutated compared with general pigs, but the structure does not change significantly. Bama-miniature pig TLR2 gene sequence has high homology compared with mice(73.4%), dog(82.4%), chicken(59.5%), cattle(85.3%), sheep(84.6%) and human(82.4%). The structure of extracellular region of TLR2 protein is forniciform arc, and consisted of a lot ofα-helix inside andβ-sheet outside of arc. There are four leucine-rich repeats and six N-linked glycosylation sites out of the membrane.(2)Toll-like receptor 4 gene cDNA consists of 2526 base pairs, coding 841 amino acids.The molecular weight and the isoelectric point of TLR4 protein was 96.4 kD and 6.58, respectively, and having a signal peptide. 5 bases of Toll-like receptor 4 gene in Bama-miniature pig mutated compared with general pig, but the structure does not change significantly. Bama-miniature pig TLR4 gene sequence has high homology compared with mice (71.9%), dog (81.5%), chicken (54.2%), cattle (86.4%), sheep (85.5%) and human (81.9%). The structure of extracellular region of TLR4 protein is forniciform arc, and consists of a lot ofα-helix inside andβ-sheet outside of arc. There are thirteen leucine-rich repeats and eight N-linked glycosylation sites out of the membrane.(3)TLR4-ASV mRNA was identified by RT-PCR.The putative TLR4-ASV protein was analysed by bioinformatics method, and the results showed that putative TLR4-ASV protein was a kind of truncated protein, having no signal peptide, not nonclassical secretory protein, six LRRs were deleted, having transmembrane region, and obviously truncated extracellular region.(4) The most stable house-keeping gene in vivo PBMC analysed by GeNorm and NormFinder is ACTB/RPL during heat stress in Bama-miniature pigs, followed by B2M > TBP > 18s RNA > GAPDH (from highest stability to lowest stability), but that is B2M+TBP in vitro cultured PBMC afer heat shock and the stability order is B2M+TBP > B2M> TBP> RPL> ACTB> 18sRNA> GAPDH (from highest stability to lowest stability).(5) In in vivo model, there was a first-increased and then-decreased tendency in TLR2 mRNA level of stressed groups with time. In stressed groups TLR2 mRNA level increased significantly compared TLR2 mRNA in same time from the 7th day. TLR4-All mRNA level was higher than that in control pigs at any times during heat stress in Bama-miniature pigs (P<0.01).The change of cortisol concentration in stress pigs was increased with time. Whereas, there was no significant diference in the cortisol level between control pigs and stressed pigs on the first day (P<0.05), but that increased significantly on the 7th, 14th and 21th day (P<0.05) after heat stress. There was no obvious changing tendency in IL-2 concentration of control pigs as time goes by, but that increased in stress pigs with time. The IL-2 level in stressed pigs was significantly lower than that of control pigs on the 7th day after stress (P<0.05), and the 7th day (P<0.01), then came to normal level. Significant difference was not found in stressed pigs and control pigs at any time of treatment.(6) In in vitro model, there was no obvious change in TLR2 mRNA level in control pigs. TLR2 mRNA level of stressed pig increased significantly and came to peak on 3th hour after treatment (P<0.05). There was no obvious change in TLR4-All mRNA level in control pigs with time. TLR4-All mRNA level in stressed pigs increased significantly on 3th hour (P<0.05) compared with that in control pigs, and then came to normal level. There was no obvious change in TLR4-ASV mRNA level of control pigs whereas there was a first - increased and then - decreased tendency in TLR4-ASV mRNA level of stressed pigs with time. TLR4-ASV mRNA level in stressed pigs increased significantly on 3th hour (P<0.01), and decreased on 6th and 9th hour, but still higher than that of control groups (P<0.05). IFN-γlevel in stressed pigs decreased significantly on 1th hour (P<0.05), and then came to increase, and there was no significant difference between control pigs and stressed pigs on 3th, 6th and 9th hour under post-stress recovery There was no obvious change in IL-12 level in control pigs. IL-12 level in stressed pigs began to increase on 1th hour post-stress recovery, and was significantly higher than that of control groups on 6th hour (P<0.05), then recover to normal level.The main conclusions of this study are as follows: (1) TLR2, TLR4 and TLR4-ASV mRNA were cloned successfully in this study.(2) The most suitable reference gene in PBMC of Bama-miniature pig during heat stress is B2M+TBP, whereas in in vitro cultured Bama-miniature pig PBMC, the most suitable reference gene is ACTB.(3)In in vivo model: heat stress can upregulate TLR2 and TLR4 mRNA level in PBMC of Bama - miniature pigs as well as serum cortisol concentration. Heat stress can regulate IL-2 level in Bama-miniature pig but did not influence the IL-8 level.(4)In in vitro model: heat stress can regulate TLR2, TLR4 and TLR4-ASV mRNA level as well as IL-12 concentration in in vitro cultured PBMC of Bama-miniature pig.Contrarily, heat stress can decrease IFN-γlevel significantly.
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