虾夷扇贝脂多糖诱导的肿瘤坏死因子LITAF 基因的克隆及表达分析

摘要

脂多糖诱导的肿瘤坏死因子(Lipopolysaccharide-induced TNF-alpha factor,LITAF)是一类重要的炎症细胞因子,在先天性免疫系统中发挥重要的介质作用.文章根据虾夷扇贝LITAF 基因EST 序列,应用RACE 技术克隆了虾夷扇贝LITAF 全长cDNA,对序列及编码的氨基酸进行生物信息学分析.结果显示,该基因cDNA 全长1 551 bp,其5′非编码区包含76 bp,3′非编码区包含1 001 bp; 开放阅读框(ORF)为474 bp,编码157 个氨基酸,氨基酸序列中存在一个保守的LITAF 结构域; 理论分子量16.99 kDa,等电点为6.24.LITAF 基因序列为3 698 bp,由3 个外显子和两个内含子组成.利用实时荧光定量PCR 技术分析LITAF 在虾夷扇贝不同组织、不同胚胎发育阶段以及鳗弧菌(Vibrio anguillarum)刺激后各时间段的表达情况.结果表明:LITAF 基因在所检测的6 个成体组织中均有表达,其中肾脏的表达量最高; 胚胎发育的7 个时期中,担轮幼体时期表达量最高; 菌刺激36 h实验组与对照组的表达量差异大.LITAF 基因是LITAF 家族的一员,推测LITAF 基因参与虾夷扇贝的先天性免疫反应.%The lipopolysaccharide-induced TNF-alpha factor (LITAF) is an inflammatory cytokine, which plays an important role in innate immunity system. Based on the expressed sequence tag (EST) of Japanese scallop (Mizuhopecten yessoensis), the cDNA of LITAF gene was amplified using rapid amplification of cDNA ends (RACE) approach. Results showed that the full-length cDNA of LITAF is 1 551 bp consisting of a 5'untranslated region (UTR) of76bp, a 3'UTR of 1 001 bp, and an open reading frame (ORF) of 474 bp encoding apolypeptide of 157 amino acids, and there is a conserved LITAF domain in amino acid sequences. The estimated molecular mass is 16.99 kDa and the theoretical isoelectric point is 6.24. The total length of LITAF is 3 698 bp, which includes three exons and two introns. Real-time quantitative PCR was carried out to measure LITAF mRNA expression in adult tissues and monitor mRNA expression patterns during embryonic development after bacteria (Vibrio anguillarum) challenged. The expression level of LITAF mRNA was detected in all the adult tissues with the highest in the kidneys. The trochophore owns the highest expression level of LITAF in embryonic development. LITAF expression showed significant difference(P<0.01)between the control and bacteria challenged specimens at 36 h. These results suggest that the LITAF should be a member of the LITAF family that perhaps involved in the innate immune response of Japanese scallop.