大黄鱼免疫球蛋白M重链基因全长cDNA序列的克隆与表达

Full-length cDNA Cloning and Expression of LcIgMH Gene from Larimichthys crocea

从本实验室自建的大黄鱼表达标签数据库中获取了大黄鱼免疫球蛋白M重链(LcIgMH)基因片段,并以此设计SMART-RACE引物,最终得到1917 bp的全长c DNA序列。经分析发现其含6 bp的5'非编码区(5'UTR)、176 bp的3'UTR以及1738 bp的开放阅读框(ORF);预测的ORF共编码584个氨基酸,蛋白质分子量为65. 2 ku,等电点为5. 84;利用Singal P 4. 1 Server发现其5'端含有信号肽(1-19 aa);Blast P Server的结果显示,其与红笛鲷的IgMH序列一致性最高,达到64%;利用IMGT/Domain Gap Align进行重链可变区与恒定区预测,发现含有1个重链可变区与4个重链恒定区,同时,预测还发现可变区含有典型的3个高变区与4个骨架区结构。实时荧光定量PCR结果显示该基因在头肾与脾脏中大量表达,并显著高于其他各组织,同时雌雄鱼LcIgMH在各组织中表达量均无显著性差异,表明LcIgMH表达与性别无关。注射副溶血弧菌进行感染4 d后,脾脏内LcIgMH表达量最高,并且与对照组之间存在显著性差异(P <0. 05),但感染后第8天的表达水平已与对照组无显著性差异(P> 0. 05),显示LcIgMH与大黄鱼的免疫应答反应相关。

Based on a fragment of the immunoglobulin heavy chain(LcIgMH)gene from Larimichthys crocea expressed sequence tags database of our lab,a full-length LcIgMH cDNA was obtained by the SMART-RACE.The full-length cDNA is1917bp,containing a6bp5'untranslated region(5'UTR),a176bp3'UTR,and a1738bp open reading frame(ORF)which encodes584amino acids.The relative molecular weight of the protein is65.2ku,and its isoelectric point is5.84.A signal peptide(1-19aa)was found in its N terminal sequence by SingalP4.1Server.The BlastP result showed that the sequence identity between LcIgMH and Lutjanus sanguineus IgMH was the highest,reaching64%.A heavy chain variable region and4heavy chain constant regions were found by the IMGT/DomainGapAlign Server.The predicted variable region contains typical3hypervariable regions and4framework regions.The expression of LcIgMH in tissues was detected by quantitative real-time PCR(qRT-PCR).The results show that LcIgMH is extremely highly expressed in the head kidney and spleen,while almost no expression in other tissues.There is no significant difference of LcIgM expression between tissues from male and female,indicating that the expression of LcIgM is not related to gender.After4days of infection with Vibrio parahaemolyticus,the expression level of LcIgMH in spleen was significantly increased compared to the control group(P<0.05).However,the expression level of the experi-mental group and control group had no significant difference at8days after injection.