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.Vibrio harveyi is a kind of conditioned pathogen often 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 cloned into a pMD 18-T vector.After sequencing and analyzing,the FlaA gene was found to contain 1 140 bp,which encodes 379 deduced amino acids.Presumably the FlaA gene would encode a protein of 40.6 kDa based on DNA-deduced amino acid sequence.When compared with homologs in other vibrios from Genbank,the FlaA gene of V.harveyi revealed the greatest homology with that of V.cholerae(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 flagellin.Then a pair of specific primers,with a short nucleotide sequence encoding Flag tag,were designed according to the obtained sequence of the FlaA genes to amplify the FlaA of V.harveyi.The PCR product was cloned into eukaryotic expression vector pcDNA3.1(+)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 flagellin in V.harveyi was constructed,which would be a foundation for further study on its DNA vaccine.国家863项目(2003AA603011);; 福建省重大科技项目(2002N009
