哈维氏弧菌FlaA基因的克隆、序列分析及真核表达质粒的构建
Cloning and sequencing of FlaA gene of Vibrio harveyi and construction of its eukaryotic expression recombinant plasmid
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摘要: 哈维氏弧菌是水产病害中常见的致病菌.参照基因库上登录的弧菌鞭毛丝蛋白FlaA基因序列设计简并引物,PCR扩增哈维氏弧菌TS-628株的FlaA全基因,并克隆到pMD 18-T载体中测序,经序列分析该基因全长1 140 bp,编码379个氨基酸.与基因库中其他弧菌的同源基因序列比较显示,哈氏弧菌FlaA基因与霍乱弧菌FlaA基因的同源性最高(79.5%),该基因编码的多肽缺乏半胱氨酸,并在N-,C-两端的氨基酸序列较为保守,中间区域的变异较大.对该蛋白的氨基酸组成及空间结构进行了分析和预测,并推测该蛋白质的平均分子量为40.6 kDa.在该基因末端加上一段编码Flag短肽的核苷酸序列后克隆到真核表达载体pcDNA3.1(+),获得带哈氏弧菌鞭毛丝蛋白FlaA基因的真核表达重组质粒pcDNA-FlaA-tag,为其DNA疫苗的进一步研究奠定了基础.Abstract: Vibrio harveyi is a kind of conditioned pathogen of ten found in the marine fishery aquaculture. In this study, a pair of degenerate primers were designed according to the homologous sequences of the FlaA genes from Genbank to amplify the FlaA gene of V. harveyi TS-628 strain, and the suitable PCR product was clonedin to a pMD 18-T vector. After sequencing and analyzing, the FlaA gene was found to contain 1 140 bp, which encodes 379 deducedamino acids. Presumably the FlaA gene would encode a protein of 40.6 kDa based on DNA-deducedam inoacid sequence. When compared with homologs in other vibrios from Genbank, the FlaA gene of V. harveyi revealed the greatest homology with that of V. choler ae(79.5%). The comparison also indicated that these polypeptides were typically conserved in amino and carboxy-termini, while the central regions were more diverse and there were no cysteine residues in the flag ellin. Then apair of specific primers, with a short nucleotide sequence encoding Flagtag, were designed according to the obtained sequence of the FlaA genes to amplify the FlaA of V. harveyi. The PCR product was clonedinto eukaryotic expression vector pcDNA 3.1(+) and the positive clone was chosen and identified through the digestion analysis and sequencing. A eukaryotic expression recombinant plasmid, pcDNA-FlaA-tag, containing the FlaA gene of polar f lagellin in V. harveyi was constructed, which would be a foundation for fur ther study on its DNA vaccine.
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Key words:
- Vibrio harveyi /
- FlaA gene /
- gene clone /
- sequence analysis /
- eukaryotic expression recombinant plasmid
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COPLAND J W,GREY D L.Management of wild and cultured sea bass/barramundi(Lates calcarifer Bloch)[A].Proceeding of an International Workshop Help at Darw in N T,Australia[C].ACIAR Proceedings,1987.20. AOKI T,EGUSA S.Drug sensitivity of Aeromonas liquefaciens isolated from freshwater fishes[J].Bull Japan Soc Fish,1971,37(3):176-185. FUDA H,SOYANO K,YAMAZAKI F,et al.Serum immunoglobulin(IgM) during early development of masu salmon(Oncorhychus masou)[J].Comp Biochem and Physiol,1991,99A:637-643. 相建海.海水养殖生物病害发生与控制[M].北京:海洋出版社,2001. FYNAN E F,WEBSTER R G,FULLER D H,et al.DNA vaccines:A novel approach to immunization[J].Int J Immunopharmac,1995,17(2):79-83. HEPPELL J,DAVIS H L.Application of DNA vaccine technology to aquaculture[J].Advanced Drug Delivery ReviewS,2000,(43):29-43. HEPPELL J,LORENZEN N,ARMSTRONG N K,et al.Development of DNA vaccines for fish:vector design,intramuscular injection and antigen expression using viral hemorrhagic septicemia virus genes as model[J].Fish Shellfish Immunol,1998,8:271-287. 周化民,苏永全,王军,等.副溶血弧菌(Vibrio parahaemolyticus)FlaI基因的合成及其克隆与鉴定[J].自然科学进展,2002,12(1):101-103. 覃映雪,苏永全,周化民,等.副溶血弧菌极鞭毛蛋白FlaB基因的克隆与序列分析[J].农业生物技术学报,2003,11(6):656-657. 陈奖励.水产微生物学[M].北京:农业出版社,1993.474-478. DAS M,CHOPRA A K,WOOD T,et al.Cloning,sequencing and expression of the flagellin core protein and other genes encoding structural proteins of the Vibrio cholerae flagellum[J].FEMS Microbiology letters,1998,165:239-246. KIM Y K,MCCARTER L L.Analysis of the polar flagellar gene system of Vibrio parahaemolyticus[J].J Bacteriol,2000,182:3 693-3 704. MCGEE K,HORSTEDT P,Milton D L.Identification and characterization of additional flagellin genes from Vibrio anguillarum[J].J Bacteriol,1996,5 188-5 198. 覃映雪,池信才,苏永全,等.网箱养殖石斑鱼溃疡病病原的研究[J].水产学报,2004,28(3):297-302. SAMBROOK J,FITCH E,MANIATIS T.Molecular Cloning:A Laboratory Manual.2nd ed.[M].New York:Cold Spring Harbor Laboratory Press,1989. HOMMA M,FUJITA H,YAMAGUCHI S,et al.Regions of Salmonella typhimurium flagellin essential for its polymerization and excretionp[J].J Bacteriol,1987,169:291-296. KUWAJIMA G,KAWAGISHI I,HOMMA M,et al.Export of an N-terminal fragment of Escherichia coli flagellin by a flagellum-specific pathway[J].Proc Natl Acad Sci USA,1989,86:4 953-4 957. WINSTANLEY C,MORGAN A W,PICKUP R W,et al.Molecular cloning of two Pseudomonas flagellin genes and basal body structural genes[J].Microbiology,1994,140:2 019-2 031. YANG E C H,SCHRANK G D,FREEMAN B A.Purification of Flagellar Cores of Vibrio cholerae[J].J Bacteriol,1977,1 121-1 128. TOTTEN P A,LORY S.Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK[J].J Bacteriol,1990,172:7 188-7 199. MILTON D L,O'TOOLE R,HORSTEDT P,et al.Flagellin A is essential for the virulence of Vibrio anguillarum[J].J Bacteriol 1996,178(5):1 310-1 319. 覃映雪,王军,王世峰,苏永全,庄轩.重组质粒pcElaA在青石斑鱼肌肉中的表达研究.高技术通讯(待刊).
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