摘要
采用同源克隆和RACE技术扩增到鲈鱼(Lateolabrax japonicus)免疫球蛋白M(Immunoglobulin M,IgM)重链(Heavychain,H)基因全长cDNA序列。鲈鱼IgM cDNA全长为1 901 bp,开放阅读框包含1 749 bp,编码582个氨基酸。根据鲈鱼IgM和其他硬骨鱼免疫球蛋白重链恒定区的氨基酸序列构建的系统发育树表明IgM、IgZ和IgD分别聚为一枝,其中IgM与IgZ分支的进化关系较近,而与IgD分支的进化关系较远。RT-PCR检测IgM在鲈鱼各组织器官的表达情况,其中在头肾及脾脏中表达量最高,心脏、肌肉及脑中几乎不表达。利用已获得的鲈鱼IgM cDNA序列,构建原核表达载体pQE30-IgM,并在M15大肠杆菌中成功诱导表达了分子量为63kD的重组蛋白His-IgM,Western-blotting显示鲈鱼IgM重组蛋白能与鼠源抗6×His的单克隆抗体特异性结合,说明已经获得了基因工程表达IgM重链蛋白。
In the present study, the complete cDNA sequence of the heavy chain of immtmoglobulin M （IgM） in Lateolabrax japonicus was cloned using the method of homologous cloning and RACE technique. The full-length cDNA of IgM was 1,901 bp, and encoded a polypeptide of 582 amino acids. A phylogenetic tree was constructed based on the alignment of amino acid sequences from the constant regions in IgM heavy chain of L.japonicas and other related teleost fishes. The results showed that the IgM, IgZ, and IgD from various fishes clustered together, respectively, and formed three major branches, while the branches oflgM and IgZ generated from a single root suggested a closer evolutionary relationship between IgM and IgZ. RT-PCR results showed the highest IgM expression level in bead-kidney, and an almost silence expression in heart, muscle and brain. The recombinant expression plasmid （pQE30-IgM） was constructed with encoding fragment of L. japonicas IgM heavy chain cDNA and the prokaryotic expression vector （pQE30）, and transformed into Escherichia coli M15. The recombinant protein was identified by SDS-PAGE and Western blot using anti- 6xHis monoclonal antibody which was specially binding with 6~His of the fusion protein.
出处
《生态科学》
CSCD
北大核心
2013年第2期218-223,共6页
Ecological Science
基金
中央高校基本科研业务费专项资助基金(21612111)
暨南大学引进优秀人才科研启动基金(50624065)
暨南大学国家级大学生创新创业训练计划项目(1210559007)
关键词
鲈鱼
免疫球蛋白M
克隆
组织分布
原核表达
Lateolabraxjaponicus
immunoglobulin M
clone
tissue distribution
prokaryotic expression
