CLONING AND EXPRESSION OF 2 GENES ENCODING HIGHLY HOMOLOGOUS HEMOLYSINS FROM A KANAGAWA-PHENOMENON-POSITIVE VIBRIO-PARAHAEMOLYTICUS T4750 STRAIN
CLONING AND EXPRESSION OF 2 GENES ENCODING HIGHLY HOMOLOGOUS HEMOLYSINS FROM A KANAGAWA-PHENOMENON-POSITIVE VIBRIO-PARAHAEMOLYTICUS T4750 STRAIN
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DOI：
10.1016/0378-1119(90)90129-f
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发表时间：
1990-09-01
期刊：
影响因子：
3.5
通讯作者：
YAMAMOTO, K
中科院分区：
文献类型：
--
作者：
IIDA, T;YAMAMOTO, K
我们从神奈川现象阳性菌株T4750中克隆并测序了编码热稳定直接溶血素（TDH）的基因，TDH是副溶血性弧菌胃肠炎的一种可能的毒力因子。发现该菌株含有与Nishibuchi和Kaper先前报道的tdh基因同源的两个序列（tdhA和tdhS）[J. Bacteriol. 162（1985）558-564]和Taniguchi等人[Microb.帕托格1（1986）425-432]。tdhA与tdhS编码区序列同源性为97.2%。TdhA的推导的氨基酸（aa）序列（不包括推定的信号肽）与从副溶血性弧菌纯化的TDH蛋白的氨基酸（aa）序列相同[Tsunasawa et al.，J.Biochem.101（1987）111-121]，除了Glu 118代替Gln 118。尽管从第二个基因tdhS推导出的aa序列与TDH蛋白有8个残基不同，但它与从先前克隆的tdh基因推导出的Tdh序列一致。tdhA和tdhS都在大肠杆菌中表达具有生物活性的溶血素。而从副溶血弧菌T4750培养上清中纯化的TDH的表观分子大小与在大肠杆菌中合成的TdhA蛋白相同。coli，其大小大于TdhS。Western blotting检测到副溶血性弧菌T4750培养上清中只有一条带，其迁移率与纯化的TDH无明显区别。这些数据表明，tdhA是在副溶血性弧菌T4750的培养上清液中发现的TDH的结构基因，并且在所采用的测试条件下，在菌株T4750中仅存在部分（如果有的话）tdhS表达。
We have cloned and sequenced the gene encoding thermostable direct hemolysin (TDH), a possible virulence factor in Vibrio parahaemolyticus gastroenteritis, from a Kanagawa-phenomenon-positive strain, T4750. This strain was found to contain two sequences (tdhA and tdhS) homologous to the tdh gene previously reported by Nishibuchi and Kaper [J. Bacteriol. 162 (1985) 558-564] and Taniguchi et al. [Microb. Pathog. 1 (1986) 425-432]. Sequence homology of the coding region between tdhA and tdhS was 97.2%. The deduced amino acid (aa) sequence of TdhA, excluding the putative signal peptide, was identical to that of TDH protein purified from V. parahaemolyticus [Tsunasawa et al., J. Biochem. 101 (1987) 111-121] except for Glu118 instead of Gln118. Although the aa sequence deduced from the second gene, tdhS, differed in eight residues from the TDH protein, it agreed with the sequence of Tdh deduced from the previously cloned tdh gene. Both tdhA and tdhS expressed biologically active hemolysins in Escherichia coli. While the apparent molecular size of TDH purified from a culture supernatant of V. parahaemolyticus T4750 was identical to TdhA protein synthesized in E. coli, it was larger than TdhS. Only one band was detected in the culture supernatant of V. parahaemolyticus T4750 by Western blotting; its mobility was indistinguishable from that of purified TDH. These data suggest that tdhA is the structural gene for TDH found in the culture supernatant of V. parahaemolytics T4750, and that there was only partial, if any, tdhS expression in the strain T4750 under the test conditions employed.