A Full-length CDNA Cloning And Expression Analysis Of Alphal-antitypsin Gene From Grass Carp

Alphal-antitrypsin is the most abundant serine protease inhibitor in plasma,and it is also an important regulatory factor to maintain the stability of the internal environment.In higher vertebrates,the studies related to its structure,mechanism,the activity of regulation,expression,purification and other aspects has achived some progress,but there are fewer reports on it in fish.In this contex,the gene specific primers were designed based on the alphal-antitrypsin EST obtained from grass carp hepatopancreas eDNA library (CK232919) and zebrafish(AAI24742.1) to clone the full-length eDNA of alphal-antitrypsin using rapid amplification cDNA end(RACE).The sequence analysis shows that the full-length of alphal-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.Prediction analysis showed that the deduced amine acids contain some potential sites,such as:5 protein kinase C sites,3 N-glycosylation sites,7 tyrosine kinaseⅡsites,seven N-aeyl sites,a super-family serine protease Serpins signature;a possible signal peptide of 21 amino acids,and two transmembrane region,which are located in 1 to 21 and 101 to 119 amino acids respectively.Structure predictions of alphal-antitrypsin from grass carp were performed by SOPMA and Swiss-model software.The results indicated that the secondary structure of alphal-antitrypsin is composed ofα-helixes,β-turns,random coils,extended strands,andα-helix has the highest proportion;the tertiary structure have great similarity to alphal-antitrypsin from other species.To reveal the homology and evolutionary relationship of grass carp alphal-antitrypsin with those of other species,we performed amino acid sequence homology alignment using the Clustal W program and constructed unrooted phylogenic tree of alphal-antitrypsin by the neighbor-joining method,basing on the paired alignment of all amino acid sequences from different species,the analysis show that the amino acid sequence of alphal-antitrypsin from grass carp 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 alphal-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 alphal-antitrypsin may be involved in the immune response for Aeromonas hydrophila infection.