Molecular cloning, genomic structure, polymorphism analysis and recombinant expression of a α1-antitrypsin like gene from swamp eel, Monopterus albus

• Published in: Fish & shellfish immunology Vol. 62; pp. 124 - 138
• Main Authors: Li, Wei, Wang, Quanhe, Li, Shaobin, Jiang, Ao, Sun, Wenxiu
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.03.2017
• Subjects: Aeromonas hydrophila - physiology; alpha 1-Antitrypsin - chemistry; alpha 1-Antitrypsin - genetics; alpha 1-Antitrypsin - metabolism; Amino Acid Sequence; Animals; Apolipoprotein A-I - genetics; Apolipoprotein A-I - metabolism; Base Sequence; Cloning, Molecular; DNA, Complementary - genetics; DNA, Complementary - metabolism; Escherichia coli - genetics; Fish Diseases - genetics; Fish Diseases - immunology; Fish Diseases - microbiology; Fish Proteins - chemistry; Fish Proteins - genetics; Fish Proteins - metabolism; Gene Expression Regulation; Genomic structure; Gram-Negative Bacterial Infections - genetics; Gram-Negative Bacterial Infections - immunology; Gram-Negative Bacterial Infections - microbiology; Gram-Negative Bacterial Infections - veterinary; Immunity, Innate - genetics; NF-kappa B - genetics; NF-kappa B - metabolism; Phylogeny; Polymorphism; Recombinant expression; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Alignment - veterinary; Smegmamorpha - genetics; Swamp eel; Transferrin - genetics; Transferrin - metabolism; α1-antitrypsin; Recombinant expression; α1-antitrypsin; Genomic structure; Swamp eel; Polymorphism
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2017.01.021

Alpha-1-antitrypsin (AAT) is a highly polymorphic glycoprotein antiprotease, involved in the regulation of human immune response. Beyond some genomic characterization and a few protein characterizations, the function of teleost AAT remains uncertain. In this study we cloned an AAT-like gene from a swamp eel liver identifying four exons and three introns, and the full-length cDNA. The elucidated swamp eel AAT amino acid sequence showed high homology with known AATs from other teleosts. The swamp eel AAT was examined both in ten healthy tissues and in four bacterially-stimulated tissues resulting in up-regulation of swamp eel AAT at different times. Swamp eel AAT transcripts were ubiquitously but unevenly expressed in ten tissues. Further, the mature peptide sequence of swamp eel AAT was subcloned and transformed into E. coli with the recombinant proteins successfully inhibiting bovine trypsin activity. Analysis of recombinant AAT showed equimolar formation of irreversible complexes with proteinases, high stability at pH 7.0–10.0 and temperatures below 55 °C. Serum AAT protein level significantly increased in response to inflammation with AAT anti-sera, and, NF-κB, apolipoprotein A1 and transferrin gene expression were dramatically decreased over 72 h post recombinant AAT injection. Lastly, examination of swamp eel AAT allelic polymorphism identified all alleles in both healthy and diseased stock except allele*g, found only in diseased stock, but without statistical difference between the distribution frequency of allele*g in the two stocks. These results are crucial to our ongoing study of the role of teleost AAT in the innate immune system. •Genomic structure of swamp eel AAT was determined and showing a great similarity to that of human AAT.•Recombinant swamp eel AAT exhibited similar biochemical characteristics to known AATs.•Serum AAT level was significantly upregulated after bacterial stimulation.•Intraperitoneal injection of the AAT protein decreased the expression of NF-κB, apolipoprotein A1, and transferrin gene.•Distribution frequencies of almost AAT alleles displayed no significant differences in sick and healthy individuals.