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为探究中华鳖天然免疫的分子机制,针对TLR2(Toll-like receptor 2)保守区设计兼并引物,从中华鳖脾cDNA中扩增出了740bp的目标序列。测序和序列分析结果表明,该序列包含富含半胱氨酸型C端结构域、跨膜结构域和TIR结构域;与斑马鱼、鸡等脊椎动物相应的TLR2序列相似性为60.5%～74.4%。采用荧光定量PCR检测中华鳖感染嗜水气单胞菌后各组织TLR2mRNA表达的变化,结果显示,感染后第24小时,中华鳖脾和外周血TLR2mRNA相对表达水平升高,其中外周血中的增幅最显著,是对照组的8.57倍;中华鳖外周血经4μg/mL酵母聚糖刺激后,第2小时TLR2mRNA相对表达水平较高。首次获得了中华鳖TLR2部分cDNA序列,并分析了不同刺激物对中华鳖TLR2mRNA转录的影响。
In order to explore the molecular mechanism of innate immunity in soft-shelled turtle Trionyx sinensis, a target sequence of 740bp was amplified from the spleen cDNA of Chinese soft-shelled turtle according to the degenerated Toll-like receptor 2 (TLR2) conserved region. Sequence analysis and sequence analysis showed that the sequence contained a Cysteine-rich C-terminal domain, a transmembrane domain and a TIR domain. The TLR2 sequence similarity of vertebrates such as zebrafish and chicken was 60.5% -74.4 %. Fluorescent quantitative PCR was used to detect TLR2 mRNA expression in each tissue of Aeromonas hydrophila infected with Aeromonas hydrophila. The results showed that the relative expression level of TLR2 mRNA in the spleen and peripheral blood of Trionyx sinensis increased at 24 hours after infection, and the increase in peripheral blood The most significant was 8.57 times that of the control group. The relative expression of TLR2 mRNA in the second hour after the stimulation of 4μg / mL Zymosan in the turtle’s peripheral blood was higher. The partial cDNA sequence of TLR2 in Chinese soft-shelled turtle was obtained for the first time and the effects of different stimuli on TLR2 mRNA transcription in soft-shelled Turtle (Trionyx sinensis) were analyzed.