鲈鱼白细胞介素-8 cDNA序列的克隆与原核表达

采用RT-PCR方法扩增和克隆了鲈鱼白细胞介素-8(LjIL-8)的编码阅读框。该编码阅读框由300个核苷酸组成,编码由99个氨基酸组成前体蛋白。LjIL-8氨基酸序列与哺乳动物和鱼类IL-8类似物的氨基酸同源性分别为23%~48%和25%~92%。氨基酸序列分析表明,N端存在一长23个氨基酸的信号肽,含有形成2个链内二硫键的4个半胱氨酸。分子进化分析表明,LjIL-8与欧洲鲈鱼的亲缘关系最近。将LjIL-8编码序列克隆到原核表达载体pET-28a (+),转化E.coli BL21(DE3)后用IPTG进行诱导表达,SDS-PAGE检测结果表明,预期分子量大小的小分子蛋白在大肠杆菌中成功表达,Western-blotting检测结果显示,目的蛋白能与抗6×His单克隆抗体发生特异性反应。

Cloning and prokaryotic expression of Lateolabrax japonica interleukin-8 cDNA

cDNA encoding Lateolabrax japonica interleukin-8 (LjIL-8) open reading frame (ORF) was cloned by RT-PCR. Sequencing results showed that the LjIL-8 ORF nucleotide sequence was 300 bp in length, encoding a prepropeptide of 99 amino acids. The deduced mature amino acid sequence of LjIL-8 was compared with those of several fish and mammalian species. The results showed that the LjIL-8 had 23%-48% and 25%-92% identities with mammalian and fish IL-8s in deduced amino acid sequence. Phylogenetic analysis showed that the LjIL-8 had close relationship with Dicentrarchus labrax IL-8. Recombinant expression plasmid of pET-28a (+) was constructed by inserting the LjIL-8 ORF sequence into the prokaryotic expression vector pET-28a (+) . An expected protein band was observed on SDS-PAGE gel, recognized by monoclonal antibody against 6×His in Western-blotting assay.