斑节对虾溶菌酶基因克隆及序列分析
ISOLATION AND CHARACTERIZATION OF THE LYSOZYME-ENCODING GENE FROM THE BLACK TIGER SHRIMP,PENAEUS MONODON
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摘要: 参考对虾溶菌酶基因和类溶菌酶基因及其他多种生物的溶菌酶基因序列,设计并合成引物.运用RT-PCR技术,从斑节对虾血细胞总RNA中扩增获得特异性片段.所获片段回收纯化后克隆到pGEM-T Easy Vector系统的T载体上.重组子的序列分析表明,所克隆的斑节对虾溶菌酶基因片段长68bp,包括溶菌酶基因开放阅读框(ORF)477bp和3′端非编码区的181bp.477bp ORF共编码18个氨基酸,包括溶菌酶成熟肽140个氨基酸残基和信号肽18个氨基酸残基.斑节对虾溶菌酶成熟肽推测分子量为16,32kd,等电点为8.78.与南美白对虾溶菌酶基因的碱基序列及推测氨基酸序列比较,同源性分别为89.%和93.0%;与日本对虾类溶菌酶基因的同源性分别为84.0%和91.0%.进一步的序列分析表明,斑节对虾溶菌酶氨基酸序列与多种类群生物的 c-型溶菌酶氨基酸序列具有较高的同源性,并具有与c-型溶菌酶相同的活性位点氨基酸残基 Glu51和 Asp68,且与活性位点相邻的序列高度保守.斑节对虾溶菌酶氨基酸序列还具有与c-型溶菌酶相同的结构氨基酸--8个半胱氨酸残基.因而可认为所克隆的斑节对虾溶菌酶基因属c-型溶菌酶基因.Abstract: Lysozyme is an enzyme which acts as a hydrolase to break down the Beta-(1,-4)linkages in the peptidoglycan layer of bacterial cell walls.Bacteria is either destroyed directly or is opsonized,enabling its destruction through phagocytosis.Researches showed that lysozyme is also an important immune factor in shrimps.Lysozyme activity has been considered as one of the basic biochemical characters to estimate the immune state of shrimps.In order to study the immune function of lysozyme in shrimps,it is necessary to isolation the shrimp lysozyme gene.Lysozyme gene of Penaeus monodon was cloned by RT-PCR (reverse transcription PCR).The primer was designed referring to the lysozyme gene and lysozyme-like gene sequences of shrimps and other species.High quality total RNA was isolated from hemocytes of Penaeus monodon. The first strand cDNAs encoding lysozyme were obtained by using total RNA as templates and then amplified.A specific band about 700bp in length in PCR products was showed by gel electrophoresis.After purified the amplified cDNA fragments were inserted into pGEM-T Easy vector and sequenced.Sequencing result showed that the total length of the cDNA is 658bp,containing an open reading frame (ORF) of 477bp and a 181bp 3′-untranslated region.Of the 477bp ORF sequence,421bp encode 140 amino acid residues of the mature peptide and 54bp for 18 amino acid residues of the signal peptide.The estimated molecular mass of the mature peptide (140 amino acids)is 16,32kD and pI is 8.78.Alignment analyses showed that the similarities of nucleotide sequences and deduced amino acid sequences of lysozyme between P.monodon and Litopenaeus vannamei are 89.5%and 93.5%,and that are 84.0%and 91.0%between P. monodon’s and Marsupenaeus japonicus’s.Further analysis showed that the lysozyme from P. monodon possesses high homology with that of chicken-type lysozyme of other species.The lysozyme from P.monodon has the conserved Glu51 Asp68 catalytic sites,as well as the eight structural cysteine residues,which are highly conserved across many species of chicken-type lysozymes.In addition,the sequences around the catalytic site are quite conserved too.Therefore the cloned P. monodon lysozyme is presumed to be chichen-type lysozyme.
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Keywords:
- Penaeus monodon /
- Lysozyme /
- Molecular cloning /
- Characterization
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G,Mattheqs B W.The refined structure of goose lysozyme and its complex with a bound Trisaccharide [J] J.Mol.Biol.1995,245 :54-68[17] Funahashi J,Takano K,Yamagata Y,et al.Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability [J].J.Biol.Che,2002,277 (24):21792-21880[18] Shen T,Wang J Y.Biochemistry [M].Beijing∶Higher Education Publishing company.1998,266-272 [沈同,王镜岩.生物化学.北京:高等教育出版社.1998,266-281][19] Feng L,Ziling Wen.Cloning and expression ppattern of the lysozyme C gene in zebrafish [J].Mechanisms of Development,2002, 11:369-372[20] Soushi F,Izumi T T,Keiko K,et al.Protein purification,cDNA cloning and gene expression of lysozyme from eri-silkworm,Samia Cynthia ricini [J].Comparative Biochemistry and Physiology Part B.2001.128 :709-718[21] Lee W J,Paul T B.Isolation and characterization of the lysozyme-encoding gene from the silkworm Bombyx mori [J].Gene,1995, 161: 199-203 Rong X H,Ling F X.The study of the development of lysozyme [J].Chinese Journal of Biochemical Pharmaceutics,1999,20 (6):319-320 [荣晓花,凌沛学.溶菌酶的研究进展 [J].中国生化药物杂志,1999,20 (6):319-320][2] Xie Z X,Liu Q,Chen F,et al.Effect of activity on chicken lysozyme by hot negative electric-charge [J].Chinese Journal of Immunology,1997,13:148-149 [谢秩勋,刘雄,陈奉等.热负电荷对血清溶菌酶活性的影响 [J].中国免疫学杂志,1997,13:148-149][3] Sonomi M,Jun-ichi H,Ikuo H,et al.Expression of Japanese flounder c-type lysozyme cDNA in insect cells [J].Dev.Comp.Immunol.2001, 25:439-445[4] Lou S X,Mao D L.The study of the development of lysozyme [J].Chinese Tumour Clinic.1994,21 (9):709-711 [楼善贤,苗德林.溶菌酶的研究进展 [J].中国肿瘤临床,1994,21 (9):709-711][5] Chipman D M,Sharon N.Mechaniam of lysozyme action [J].Science,1969,165:454[6] Zhu Q,Chen Y.Lysozyme and its application [J].Journal of Biology,1998,33 (10):9-10 [朱奇,陈彦.溶菌酶及其应用 [J].生物学通报,1998,33 (10):9-10][7] Wang J P,Wu Q F.The current study of hemocytes and humoral immunity [J].Journal of Zhejiang Ocean University (Natural Science),2000,19 (4):354-360 [王建平,吴雄飞.虾类血细胞及体液免疫的研究现状 [J].浙江海洋学院学报(自然科学版),2000, 19 (4):354-360][8] Munoz M F,Vandenbulcke D,Saulnier et al.Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp [J].Eur J Biochem,2002,269:2678-2689[9] Alabi A O,Latchford J W,Jones D A.Demonstration of residual antibacterial activitv in plasm of vaccinated Penaeus vannamei [J].Aquaculture,2000,187:15-34[10] Liu H,Li G Y.The study of the effects of immunopolysaccharide on cultured white shrimp [J].Oceanologia et Limnologlia Sinica [J], 1998,29 (2):113-118[刘恒,李光友.免疫多糖对养殖南美白对虾作用的研究 [J].海洋与湖沼,1998,29 (2):113-118][11] Liu S Q,Jiang X L,Mou H J,et al.Effects of immunopolysaccharide on LSZ,ALP,ACP and POD activity of Penaeus Chinesis serum [J].Oceanologia et Limnologlia Sinica,1999,30(3):278-283[刘树青,江晓路,牟海津,等.免疫多糖对中国对虾血清溶菌酶、磷酸酶和过氧化物酶的作用 [J].海洋与湖沼,1999,30(3):278-283][12] Bachere E,Mialhe E,Rodriguez J.Identification of defence effectors in the haemolymph of crustaceans with particular reference to the shrimp Penaeus japonicus (Bate):prospects and applications [J].Fish Shellfish Immunol,1995,5 :597-612[13] Huang X H,Li C L,Liu C W,et al.Study on two microalgae improving environment of shrimp pond and strengthening anti-disease ability of Penaeus vannamei.Acta Hydrobiologica Sinaca.2002,26 (4):343-347 [黄翔鹄,李长玲,刘楚吾,等.两种微藻改善虾池土环境增强凡纳对虾抗病力的研究.水生生物学报,2002,26 (4):343-347][14] Gross P S,Bartlett T C,Browdy C L,et al.Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the Pacific White Shrimp,Litopenaeus vannamei,and the Atlantic White Shrimp,L.setiferus [J].Developmental and Comparative Immunology,2001,25 :565-577[15] Jun I H,Sonomi M,Ikuo H,et al.Molecular cloning,expression and evolution of the Japnanese flounder goose-type lysozyme gene,and the lytic activity of its recombinant protein [J].Biochimica et Biophysica Acta.2001,1520:35-44[16] Weaver L H,Grütter M G,Mattheqs B W.The refined structure of goose lysozyme and its complex with a bound Trisaccharide [J] J.Mol.Biol.1995,245 :54-68[17] Funahashi J,Takano K,Yamagata Y,et al.Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability [J].J.Biol.Che,2002,277 (24):21792-21880[18] Shen T,Wang J Y.Biochemistry [M].Beijing∶Higher Education Publishing company.1998,266-272 [沈同,王镜岩.生物化学.北京:高等教育出版社.1998,266-281][19] Feng L,Ziling Wen.Cloning and expression ppattern of the lysozyme C gene in zebrafish [J].Mechanisms of Development,2002, 11:369-372[20] Soushi F,Izumi T T,Keiko K,et al.Protein purification,cDNA cloning and gene expression of lysozyme from eri-silkworm,Samia Cynthia ricini [J].Comparative Biochemistry and Physiology Part B.2001.128 :709-718[21] Lee W J,Paul T B.Isolation and characterization of the lysozyme-encoding gene from the silkworm Bombyx mori [J].Gene,1995, 161: 199-203
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