军曹鱼Igκ轻链cDNA克隆及组织表达分析

Molecular cloning and tissue expression analysis of immunoglobulin light chain cDNA from cobia Rachycentron canadium Linnaeus

应用同源克隆和RACE技术克隆了军曹鱼(Rachycentron canadium Linnaeus)Igκ轻链的cDNA全序列,并分析其在组织中的表达。军曹鱼Igκ的cDNA全长969 bp,3′端的非编码区域(UTR)为188 bp,5′UTR为52 bp,开放阅读框为729 bp,编码242个氨基酸,分子量约为26.255 kD,理论等电点7.52。推测的军曹鱼Igκ恒定区氨基酸序列同(Seriola quinqueradiata)、大西洋鲑(Salmo salar)的Ig轻链同源性最高,达77%以上,可变区氨基酸序列与的相应序列同源性最高,达87%。通过构建系统进化树可以看出,军曹鱼Igκ恒定区氨基酸序列同、大西洋鲑的L3、斑点叉尾(Ictalurus punctatus)的G等鱼的轻链均为κ型的聚为一支,同大西洋鲑的L2、斑马鱼(Danio rerios)的L2、鲤(Cyprinus carpio)的L2等均为λ型的明显不同,由此可推断此序列属于Igκ型。利用半定量PCR技术,发现在健康鱼体中κ链基因在肝脏和鳃中的表达量较高,在肠和脑组织中几乎没有表达。经鲨鱼弧菌(Vibrio carchariae)JT2刺激192 h后,κ链基因在采样的各个组织中均有表达,尤其在头肾、肠、鳃和脾中的表达量较正常水平明显升高,而肝的表达量有所下降,脑组织有κ链基因的少量表达。说明经刺激后头肾、脾脏、肠、鳃是免疫球蛋白的主要表达场所,在抵御病原感染过程中发挥着重要作用。

The technique of homologous cloning and Rapid Amplification of cDNA Ends（RACE） was used to am-plify full length cDNA gene of immunoglobulin light chain（κ chain） from cobia（Rachycentron canadium Lin-naeus）.The full length cDNA of κ in cobia is 969 bp,containing a 3′ untranslated region（UTR） of 188 bp,a 5′ UTR of 52 bp,and an open reading frame（ORF） of 729 bp,encoding 242 amine acids.The estimated molecular weight of Ig κ is 26.255 kD,and the theoretical isoelectric point is 7.52.The deduced Igκ amino acid sequences of cobia were compared with those of other teleost species.For the constant region of Igκ,higher percentage similar-ity was obtained from comparisons between R.canadium and Seriola quinqueradiata and between R.canadium and Salmo salar,which was higher than 77%.For the variable region,higher percentage similarity was obtained from comparisons between R.canadium and S.quinqueradiata,which reached 87%.By the phylogenetic tree of immunoglobulin light chain constant region,Igκ amino acid sequences of cobia were clustered with S.quinquera-diata（1,2,3） and Ictalurus punctatus G chain which belong to the type of κ chain,so IgL of cobia was supposed to type of κ.Salmo salar L2 chain,Danio rerios L2 chain,Cyprinus carpio L2 chain that belong to the type of λ chain were clustered together.The expression of Igκ gene in healthy cobia was initially measured by semi-quantitative RT-PCR.It was found that the expression of the Igκ existed more obviously in liver and gill than in other tissues,but they were hardly expressed in intestine and brain.The expression of the target gene in head kidney,spleen,intestine and gill increased obviously after cobia was immunized by intraperitoneal injection with Vibrio carchariae strain JT2,while the expression in liver decreased.The result indicated that head kidney,spleen,intestine and gill are main organs for Igκ production after stimulation,and play a critical role in host-pathogen in-teraction.