[Cloning and expression of Shiga-like toxin type II variant B gene of E. coli].
A structure sequence and a DNA fragment including the signal peptide sequence and structure sequence of Shiga-like toxin II variant B subunit gene were amplified from E. coli strain O138 by PCR. After digested with restriction endonuclease EcoRI and BamHI, the two genes were orientally inserted into the polycloning site of expression vector pYA3334 (asd+) respectively. Recombinant plasmids pB0 and pB1 were constructed and amplified in E. coli X6212 (asd-). pB0 and pB1 were then introduced into avirulent Salmonella typhimurium vaccine strain X4550 (asd-) by serial transformation through intermediate strain X3730 (asd-) to construct recombinant SLT-IIvB strain. Results of nucleotide sequencing of the cloned fragments in pB0 and pB1 revealed that they were in correct ORF of SLT-IIvB. The results of SDS-PAGE and Western-blot showed that 7.6 kD protein of SLT-IIvB antigen was expressed at pretty high level in recombinant strain X4550(pB0). The results of mice immunization indicated X4550(pB0) could initiate the host to produce specific antibodies to SLT-IIvB and LPS-O antigen of X4550. So the recombinant strain X4550 (pB0) is worth considering as a candidate vaccine strain against porcine edema disease and Salmonella typhimurium infections.
Ni
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Department of Veterinary Science, College of Animal Husbandry & Veterinary Medicine, Yangzhou University, Yangzhou 225009.
Jiao
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Gao
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Zhang
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Liu
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D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012486:Salmonella typhimurium / Q000276:immunology*; D022641:Shiga Toxin 2 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D014169:Transformation, Bacterial; D014614:Vaccines, Synthetic / Q000276:immunology
D011994:Recombinant Proteins; D022641:Shiga Toxin 2; D014614:Vaccines, Synthetic