CLONING, EXPRESSION, AND SEQUENCE-ANALYSIS OF A CYTOLYTIC ENTEROTOXIN GENE FROM AEROMONAS-HYDROPHILA
CLONING, EXPRESSION, AND SEQUENCE-ANALYSIS OF A CYTOLYTIC ENTEROTOXIN GENE FROM AEROMONAS-HYDROPHILA
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DOI：
10.1139/m93-073
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发表时间：
1993-05-01
影响因子：
2.8
通讯作者：
JIN, GF
中科院分区：
文献类型：
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作者：
CHOPRA, AK;HOUSTON, CW;JIN, GF
克隆了嗜水气单胞菌腹泻分离株SSU的溶细胞性肠毒素的结构基因和调控元件，并测定了其DNA序列。以气单胞菌溶细胞性肠毒素前10个氨基末端氨基酸残基为基础的互补、混合合成寡核苷酸为探针，筛选构建于噬菌体EMBL3中的基因组文库。含有溶细胞型肠毒素基因的大肠杆菌细胞裂解物(LambdaCH4)能裂解兔红细胞，破坏中国仓鼠卵巢细胞，引起大鼠回肠分泌液，静脉注射致小鼠死亡。与溶细胞性肠毒素相关的所有生物学活性均可被兔同源多克隆抗体中和。十二烷基硫酸钠聚丙烯酰胺凝胶电泳和随后的Western印迹分析表明，利用针对细胞溶解肠毒素的抗血清或针对该毒素的合成肽产生的抗体，大肠杆菌(LambdaCH4)的细胞裂解产物显示出一条约52 kDa的蛋白条带。对一个2.8kb的Sali-BamHI片段的DNA序列分析表明，该片段有一个大的开放阅读框(1479bp)，编码54.5 kDa的蛋白质，是溶细胞性肠毒素的前体形式，含有23个氨基酸的前导肽。尽管我们的溶细胞性肠毒素与气单胞菌的两种气溶素在DNA和氨基酸水平上有很大的同源性，但这三种毒素基因的限制性内切酶图谱存在显著的差异。同样，已报道的溶气素和我们的溶细胞性肠毒素的结构基因上游的DNA序列也有相当大的差异，这表明这些结构相似的毒素分子可能受到不同的调控。最后，我们的数据显示，来自嗜水气单胞菌腹泻分离株SSU的溶细胞性肠毒素表现出与已报道的溶气素相比独特的特征。
The structural gene and regulatory element for a cytolytic enterotoxin of a diarrheal isolate, SSU, of Aeromonas hydrophila was cloned and its DNA sequence was determined. A complementary, mixed synthetic oligonucleotide based on the first 10 NH2-terminal amino acid residues of the Aeromonas cytolytic enterotoxin was used as a probe to screen a genomic library constructed in bacteriophage EMBL3. Cell lysates of Escherichia coli (lambdaCH4), containing the cytolytic enterotoxin gene, lysed rabbit red blood cells and destroyed Chinese hamster ovary cells, caused fluid secretion in rat ileal loops, and were lethal to mice when injected intravenously. All biological activities associated with the cytolytic enterotoxin were neutralized by rabbit homologous polyclonal antibodies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot analysis of the cell lysate of E. coli (lambdaCH4) revealed a protein band of approximately 52 kDa, using antisera to the cytolytic enterotoxin or antibodies generated against a synthetic peptide to the toxin. DNA sequence analysis of a 2.8-kb SalI-BamHI fragment revealed the presence of one large open reading frame (1479 bp) that would encode a protein of 54.5 kDa, a precursor form of the cytolytic enterotoxin, with a 23 amino acid leader peptide. Despite a significant amount of homology at the DNA and amino acid levels between our cytolytic enterotoxin and two aerolysins of Aeromonas species, variation in the restriction maps of these three toxin genes was prominent. Likewise, considerable divergence in DNA sequence was observed upstream of the structural genes for the reported aerolysins and our cytolytic enterotoxin, suggesting that these structurally similar toxin molecules may be regulated differently. Finally, our data showed that the cytolytic enterotoxin from a diarrheal isolate, SSU, of A. hydrophila exhibited characteristics that were unique compared with those of the reported aerolysins.