鲢IL-8基因的克隆与表达分析

采用RACE-PCR方法,从鲢(Hypophthalmichthys molitrix)头肾SMARTcDNA中获得了679 bp IL-8 cDNA序列。结果显示:该序列包含169 bp的5′非编码区,213 bp 3′端非编码区和297 bp的开放阅读框;鲢IL-8的开放阅读框编码98个氨基酸残基,其中包含构成两对二硫键的4个保守半胱氨酸。RT-PCR结果显示鲢:IL-8 mRNA主要在胰、肝脏和头肾中表达。将鲢IL-8不含信号肽的成熟多肽克隆到原核融合表达载体pQE30上,转入大肠杆菌M15内进行表达,经SDS-PAGE电泳后显示,重组融合蛋白在分子量约11kD处有一明显表达带,与预期计算的分子量大小相一致。

Gene cloning and expression of Interleukin-8(IL-8) from silver carp(Hypophthalmichthys molitrix)

Interleukin-8(IL-8) is a CXC chemokine,which can attract and activate specific types of leukocytes,such as lymphocytes and neut-rophils.The cDNA of silver carp IL-8 consists of 679 bp with 169 bp 5′ untranslated region(UTR),213 bp 3′UTR.An open reading frame of 297 bp encoded a 98-amino acid peptide,with four cysteines.Phylogenetic analyses clearly demonstrated that silver carp IL-8 resembles the IL-8s of other fish.RT-PCR results revealed that stronger products in the pancreas,liver and head kidney were detected.Then the mature peptide of silver carp IL-8 was directionally connected the expression vector pQE30 to construct pQE30-IL8 recombinant.This recombinant was transformed into E.coli M15.An expected protein about 11kD was found in M15 cell protein after inducting with IPTG by SDS-PAGE analysis.