草鱼α1-抗胰蛋白酶基因cDNA全长克隆与表达分析

A full-length cDNA Cloning and Expression Analysis ofα1-antitrypsin Gene in Grass Carp

采用RACE技术获得α1-抗胰蛋白酶基因cDNA全长序列为1 469 bp,开放阅读框为1 329 bp,可编码442个氨基酸。5′非编码区长19 bp,3′非编码区长121 bp。核苷酸序列分析表明,在N端可能存在一个由1～21位氨基酸残基组成的信号肽;与斑马鱼的同源性最好,其次是虹鳟;在系统进化上,与在斑马鱼、虹鳟共聚为一个大支。用半定量RT-PCR分析正常及细菌诱导下草鱼α1-抗胰蛋白酶基因在不同组织中的表达分布。结果显示:正常情况下,草鱼α1-抗胰蛋白酶在肝脏表达最丰富,在脾脏、前肾、前肠、中肠、后肠和也有少量表达;细菌诱导下,肝脏中表达最强,前肾、脾脏、肠道中表达均明显提高,心脏和后肾中也出现较高表达。提示α1-抗胰蛋白酶可能参与了机体对嗜水气单胞菌感染的免疫应答。

In this contex,we clone the full-length cDNA of alpha1-antitrypsin using rapid amplification cDNA end (RACE).The sequence analysis shows that the full-length of alpha1-antitrypsin cDNA from grass carp was 1469 bp,which contained a 1329 bp of open reading frame(ORF)encoding a alphal-antitrypsin precursor of 442 amino acid,flanked by a 19 bp of 5'-untranslated region(UTR)and a 121 bp of 3'-UTR;the analysis show that the amino acid sequence of alpha1 -antitrypsin from grass carp contain a possible signal peptide of 21 amino acids(1～21),and has a high similarity to that of zebrafish,followed by rainbow trout;In the evolutionary tree,the grass carp was clustered with zebrafish and rain bow.The expression of alpha1-antitrypsin gene in different tissues from bacteria-induced or health grass carp were detected with semi-quantitative RT-PCR.The results indicated that:(1)Alphal-antitrypsin is expressed in abundance in the liver in normal tissues,and there was a little expression in former kidney,former intestinal,mid-intestinal,latter intestinal; (2)The tissue-related expression was induced by bacteria showed that the mRNA level was strongestly expressed in the liver and is significantly increased in spleen,heart,former,latter kidney,former intestinal,mid-intestinal,intestinal, especially a high expression was found in heart and latter kidney.The results indicated that alpha1-antitrypsin may be involved in the immune response for Aeromonas hydrophila infection.