Molecular cloning, expression and characterization of Bmserpin-2 gene from Bombyx mori

• Autorzy: Pan Y. , Xia H. , Lu P. , Chen K. , Yao Q. , Chen H. , Gao L. , He Y. , Wang L.
• Języki publikacji: EN

Abstrakty

EN

Serpins are a broadly distributed family of protease inhibitors. In this study, the gene encoding Bombyx mori serpin-2 (Bmserpin-2) was cloned and expressed in E. coli. The Bmserpin-2 cDNA contains a 1125 bp open reading frame (ORF). The deduced protein has 374 amino-acid residues, contains a conserved SERPIN domain and shares extensive homology with other invertebrate serpins. RT-PCR analysis showed that Bmserpin-2 was expressed in all developmental stages of B. mori larvae and various larval tissues. Subcellular localization analysis indicated that Bmserpin-2 protein was located in the cytoplasm. Interestingly, real-time quantitative PCR revealed that the expression of Bmserpin-2 in the midgut of susceptible B. mori strain 306 significantly increased at 72 hours post inoculation (hpi) when infected with BmNPV. However, there was no significant increase of the Bmserpin-2 expression in resistant strain NB infected with BmNPV. Thus, our data indicates that Bmserpin-2 may be involved in B. mori antiviral response.

Słowa kluczowe

EN

gene expression; serine protease inhibitor; molecular cloning; serpin; Bmserpin-2 gene; subcellular location; bioinformatics; Bombyx mori; serpin-2

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2009
• Tom: 56
• Numer: 4
• Opis fizyczny: p.671-677,fig.,ref.

Twórcy

autor

Pan Y.

• Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, P.R.China

autor

Xia H.

autor

Lu P.

autor

Chen K.

autor

Yao Q.

autor

Chen H.

autor

Gao L.

autor

He Y.

autor

Wang L.

Bibliografia

• Abraham EG, Pinto SB, Ghosh A, Vanlandingham DL, Budd A, Higgs S, Kafatos FC, Jacobs-Lorena M, Michel K (2005) An immune-responsive serpin, SRPN6, mediates mosquito defense against malaria parasites. Proc Natl Acad Sci USA 102: 16327-16332. 
• Chamankhah M, Braun L, Visal-Shah S, O'Grady M, Baldwin D, Shi X, Hemmingsen SM, Alting-Mees M, Hegedus DD (2003) Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation. Insect Biochem Mol Biol 33: 355-369. 
• Dementiev A, Dobó J, Gettins PGW (2006) Active site distortion is sufficient for proteinase inhibition by serpins: structure of the covalent complex of α1-proteinase inhibitor with porcine pancreatic elastase. J Biol Chem 281: 3452-3457. 
• Florea L, Hartzell G, Zhang Z, Rubin GM, Miller W (1998) A computer program for aligning a cDNA sequence with a genomic DNA sequence. Genome Res 8: 967-974. 
• Gan H, Wang Y, Jiang H, Mita K, Kanost MR (2001) A bacteria-induced, intracellular serpin in granular hemocytes of Manduca sexta. Insect Biochem Mol Biol 31: 887-898. 
• Hunt LT, Dayhoff MO (1980) A surprising new protein superfamily containing ovalbumin, antithrombin-III, and α1-proteinase inhibitor. Biochem Biophys Res Commun 95: 864-871. 
• Huntington JA, Read RJ, Carrell RW (2000) Structure of a serpin-protease complex shows inhibition by deformation. Nature 407: 923-926. 
• Irving JA, Pike RN, Lesk AM, Whisstock JC (2000) Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 10: 1845-1864. 
• Jiang H, Kanost MR (1997) Characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from Manduca sexta. J Biol Chem 272: 1082-1087. 
• Kanost MR (1999) Serine proteinase inhibitors in arthropod immunity. Dev Comp Immunol 23: 291-301. 
• Khurad AM, Mahulikar A, Rathod MK, Rai MM, Kanginakudru S, Nagaraju J (2004) Vertical transmission of nucleopolyhedrovirus in the silkworm, Bombyx mori L. J Invertebr Pathol 87: 8-15. 
• Nakazawa H, Tsuneishi E, Ponnuvel KM, Furukawa S, Asaoka A, Tanaka H, Ishibashi J, Yamakawa M (2004) Antiviral activity of a serine protease from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus. Virology 321: 154-162. 
• Pike RN, Buckle AM, le Bonniec BF, Church FC (2005) Control of the coagulation system by serpins. Getting by with a little help from glycosaminoglycans. FEBS J 272: 4842-4851. 
• Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406. 
• Salzet M, Vieau D, Stefano GB (1999) Serpins: an evolutionarily conserved survival strategy. Immunol Today 20: 541-544. 
• Silverman GA, Bird PI, Carrell RW (2001) The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 276: 33293-33296. 
• Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599. 
• Tong Y, Kanost MR (2005) Manduca sexta serpin-4 and serpin-5 inhibit the prophenol oxidase activation pathway: cDNA cloning, protein expression, and characterization. J Biol Chem 280: 14923-14931. 
• Van Gent D, Sharp P, Morgan K, Kalsheker N (2003) Serpins: structure, function and molecular evolution. Int J Biochem Cell Biol 35: 1536-1547. 
• Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. Biotechniques 39: 75-85. 
• Zhu Y, Wang Y, Gorman MJ, Jiang H, Kanost MR (2003) Manduca sexta serpin-3 regulates prophenoloxidase activation in response to infection by inhibiting prophenoloxidase-activating proteinases. J Biol Chem 278: 46556-46564.