鲤鱼诱导型一氧化氮合成酶全长cDNA克隆及其在外周血白细胞中的差异表达分析

Cloning of Carp Inducible Nitric Oxide Synthase Full-length cDNA and its Differential Expression Analysis in Peripheral Blood Leukocytes

试验旨在获得鲤鱼诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)cDNA全长序列,并以此为基础探讨在丝裂原刺激下外周血白细胞中iNOS的表达变化。以从鲤鱼正常外周血白细胞cDNA文库中获得的iNOS的EST序列为基础,采用基因文库筛选和cDNA 5′末端快速扩增技术(5′-RACE)相结合的方法,成功扩增出鲤鱼iNOS cDNA全长序列,然后进行鲤鱼外周血白细胞原代培养,分成对照组和试验组,其中试验组分别为脂多糖(LPS,1.0μg/mL)刺激4、12h,刀豆蛋白A(ConA,1.0μg/mL)刺激4、24h,对照组为相同培养时间无丝裂原刺激的外周血白细胞,根据得到的iNOS cDNA全长序列和鲤鱼β-actin序列分别设计特异性引物,应用实时荧光定量PCR方法检测各组外周血白细胞中iNOS在mRNA水平上的表达情况并进行分析。序列分析结果显示,最后获得的cDNA片段共3 704bp,包含74bp的5′端非编码区,246bp的3′端非编码区,一个3 384bp的完整的开放阅读框(ORF),共编码1 127个氨基酸。序列同源性分析结果显示,该序列与鲫鱼iNOS基因同源性高达100%;实时荧光定量PCR结果显示,经LPS、ConA刺激的试验组外周血白细胞中iNOS表达量均升高,且短时间(4h)刺激的表达量高于长时间(LPS 12h;ConA 24h)刺激的表达量。综上所述,在LPS、ConA刺激过程中,iNOS在外周血白细胞中表达量均有上调,结果提示存在炎症反应的动态变化。

The study was aimed to obtain carp iNOS cDNA full-length sequence,and investigate the changes in peripheral blood leukocyte expression of iNOS in the stimulation of different mitogens.Based on EST sequences of iNOS that obtained from carp normal peripheral blood leukocytes cDNA library,full-length cDNA sequence of carp iNOS was successfully amplified by using gene library screening and rapid-amplification of cDNA 5＇ends （5＇-RACE） method.Carp peripheral blood leukocytes were divided into control and experimental groups and cultured.The experimental groups were stimulated by LPS （1.0 μg/mL） and ConA （1.0 μg/mL） for 4 and 12 h,respectively.And control groups were in the same cultured conditon time without mitogen stimulated.Real-time PCR was used to detect the expression of iNOS in peripheral blood leukocytes of each group.The results showed that the cDNA fragment was total 3 704 bp containing 74 bp 5＇ untranslated region （UTR）,246 bp 3＇UTR and a 3 384 bp ORF encoding 1 127 amino acids.Sequence homology analysis showed that the amino acid sequence of carp iNOS shared 100％ identity with Carassius auratus.After LPS and ConA stimulation,the iNOS expression of peripheral blood leukocytes in the experimental groups were increased,and the expression in the short time stimulation condition （4 h） was higher than the long time （LPS 12 h;ConA 24 h）.In conclusion,iNOS expression of peripheral blood leukocytes was raised after LPS and ConA stimulation,suggesting that there were dynamic changes in the inflammation.