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首页> 外文期刊>Infection and immunity >Cloning and expression of the leukotoxin gene of Pasteurella haemolytica A1 in Escherichia coli K-12.
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Cloning and expression of the leukotoxin gene of Pasteurella haemolytica A1 in Escherichia coli K-12.

机译：溶血巴斯德氏菌A1的白细胞毒素基因在大肠杆菌K-12中的克隆和表达。

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摘要

A clone bank of Pasteurella haemolytica A1 was constructed by partial digestion of the genomic DNA with Sau3A and ligation of 5- to 10-kilobase-pair fragments into the BamHI site of the plasmid vector pBR322. After transformation into Escherichia coli K-12, a total of 4 X 10(3) recombinant clones was obtained. These were screened for the production of P. haemolytica soluble antigens by a colony enzyme-linked immunosorbent assay blot method with a rabbit antiserum raised against the soluble antigens. The clones producing P. haemolytica soluble antigens were then analyzed for the production of the leukotoxin by a cytotoxicity assay with cells from a bovine leukemia-derived B-lymphocyte cell line as the target cells. Positive clones were identified, and subsequent restriction analysis of the recombinant plasmids showed that the same 6.3 kilobase pairs of insert DNA was cloned in either of the two orientations into the plasmid vector pBR322. One of the clones was selected for further characterization of the leukotoxin as produced in E. coli. Tests for heat lability and target cell species specificity with canine, porcine, and human peripheral blood lymphocytes indicated that the activity of the cloned leukotoxin was identical to that of the P. haemolytica leukotoxin. Furthermore, the E. coli-produced leukotoxin was also neutralized by bovine or rabbit antiserum known to have antitoxic activity. When cellular proteins from the E. coli clones were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, a 100,000-dalton protein was identified which corresponded to one of the soluble antigens found in the leukotoxic culture supernatant of P. haemolytica. These results demonstrated that the gene(s) for the P. haemolytica leukotoxin have been cloned and that the leukotoxin was expressed in E. coli.
机译：通过用Sau3A部分消化基因组DNA，并将5到10个碱基对的片段连接到质粒载体pBR322的BamHI位点，构建溶血巴斯德氏菌A1的克隆库。转化为大肠杆菌K-12后，总共获得了4个X 10（3）重组克隆。通过集落酶联免疫吸附测定印迹法，用针对可溶性抗原的兔抗血清，筛选出这些蛋白产生溶血假单胞菌的可溶性抗原。然后，以牛白血病来源的B淋巴细胞细胞系中的细胞为靶细胞，通过细胞毒性试验分析产生溶血性巴氏杆菌可溶抗原的克隆的白细胞毒素的产生。鉴定出阳性克隆，随后对重组质粒的限制性酶切分析表明，将相同的6.3kb插入片段DNA沿两个方向的任何一个克隆到质粒载体pBR322中。选择克隆之一以进一步表征大肠杆菌中产生的白细胞毒素。用犬，猪和人外周血淋巴细胞进行热不稳定性和靶细胞物种特异性的测试表明，克隆的白细胞毒素的活性与溶血性疟原虫白细胞毒素的活性相同。此外，大肠杆菌产生的白细胞毒素也被已知具有抗毒活性的牛或兔抗血清中和。当对来自大肠杆菌克隆的细胞蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹分析时，鉴定出100,000道尔顿的蛋白质，其对应于溶血毕赤酵母白毒性培养上清液中发现的一种可溶性抗原。这些结果表明，已经克隆了溶血性疟原虫白细胞毒素基因，并且该白细胞毒素在大肠杆菌中表达。

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