香鱼TGF-β1基因的克隆及其表达与鳗利斯顿氏菌感染的相关性

Cloning of sweetfish TGF-β1 and its expression following Listonella anguillarum infection

通过转录组测序的方法,获得香鱼的TGF-β1基因序列,由1134个核苷酸组成,包括一个大的开放阅读框,编码成377个氨基酸组成的前体蛋白,分子量大小为43 kDa,等电点为8.72。N端前19个氨基酸为信号肽,成熟肽由C末端112个氨基酸组成,分子量大小为12.5 kDa。序列比对表明香鱼与虹鳟同源性最高,前导肽为72%,成熟肽为93%。在健康香鱼中,TGF-β1基因在肝、脾、肾、脑、肌、肠、鳃、心组织中均有表达,在肾组织中表达量最高。鳗利斯顿氏菌侵染香鱼组织后,各组织中的TGF-β1基因mRNA表达量均显著上调,肾组织中变化最为显著,注射12h时为对照组的14.5倍。构建了原核表达质粒pET-28a-TGF-β1,并制备了多克隆抗血清,能与目的蛋白TGF-β1起特异性反应,但不与细菌蛋白自身反应。以上结果表明,香鱼TGF-β1基因表达变化与鳗利斯顿氏菌的侵染相关,揭示了TGF-β1可能在鱼类抗细菌的急性期免疫应答过程中发挥作用。

In this study,the sequence of TGF-β1 was obtained. An open reading frame is composed of 1143 nucleotides,which encoded a precursor protein consisted of 377 amino acids,and its molecular weight is 43 kDa,the isoelectric point is 8. 72. Its N-terminal19 residues are the signal peptides,and the C-terminal 112 residues are the mature peptide,whose molecular weight is 12. 5 kDa. Sequence alignment showed that sweetfish TGF-β1 shared the highest homology with rainbow trout,the leader peptide was 72%,and the mature peptide was 93%. In healthy sweetfish,TGF-β1 was expressed in the all tested tissues,and that the highest expression was in the kidney. After Listonella anguillarum infection,sweetfish TGF-β1 mRNAs were up-regulated in all detected sweetfish tissues,especially in the kidney. The prokaryotie expression vector pET-28a-TGF-β1 was constructed,and its antiserum was prepared,which can react with the target protein TGF-β1,but it can not react with the bacterial protein itself. The above results showed that sweetfish TGF-β1 expression is related with L. anguillarum infection,and it revealed that TGF-β1 may play a role in the acute phase of fish antibacterial immunity.