Guerry P, Pope P M, Burr D H, Leifer J, Joseph S W, Bourgeois A L
Enterics Program, Naval Medical Research Institute, Bethesda, Maryland 20814.
Infect Immun. 1994 Feb;62(2):426-32. doi: 10.1128/iai.62.2.426-432.1994.
Isogenic recA mutants of Campylobacter jejuni have been constructed for evaluation of their usefulness in attenuated vaccines against this major worldwide cause of diarrhea. The recA+ gene of C. jejuni 81-176 was cloned by using degenerate primers to conserved regions of other RecA proteins in a PCR. The C. jejuni recA+ gene encodes a predicted protein with an M(r) of 37,012 with high sequence similarity to other RecA proteins. The termination codon of the recA+ gene overlaps with the initiation codon of another open reading frame which encodes a predicted protein which has > 50% identity with the N terminus of the Escherichia coli enolase protein. A kanamycin resistance gene was inserted into the cloned recA+ gene in E. coli and returned to C. jejuni VC83 by natural transformation, resulting in allelic replacement of the wild-type recA gene. The resulting VC83 recA mutant displayed increased sensitivity to UV light and a defect in generalized recombination as determined by natural transformation frequencies. The mutated recA gene was amplified from VC83 recA by PCR, and the product was used to transfer the mutation by natural transformation into C. jejuni 81-176 and 81-116, resulting in isogenic recA mutants with phenotypes similar to VC83 recA. After oral feeding, strain 81-176 recA colonized rabbits at levels comparable to wild-type 81-176 and was capable of eliciting the same degree of protection as wild-type 81-176 against subsequent homologous challenge in the RITARD (removable intestinal tie adult rabbit diarrhea) model.
空肠弯曲菌的同基因recA突变体已构建完成，用于评估其在针对这种全球主要腹泻病因的减毒疫苗中的效用。通过使用简并引物对PCR中其他RecA蛋白的保守区域进行扩增，克隆了空肠弯曲菌81-176的recA⁺基因。空肠弯曲菌recA⁺基因编码一种预测的蛋白质，其分子量为37,012，与其他RecA蛋白具有高度的序列相似性。recA⁺基因的终止密码子与另一个开放阅读框的起始密码子重叠，该开放阅读框编码一种预测的蛋白质，与大肠杆菌烯醇化酶蛋白的N端具有>50%的同一性。将卡那霉素抗性基因插入大肠杆菌中克隆的recA⁺基因，并通过自然转化返回空肠弯曲菌VC83，导致野生型recA基因的等位基因替换。通过自然转化频率测定，所得的VC83 recA突变体对紫外线的敏感性增加，并且在广义重组方面存在缺陷。通过PCR从VC83 recA中扩增突变的recA基因，并将产物通过自然转化转移到空肠弯曲菌81-176和81-116中，产生具有与VC83 recA相似表型的同基因recA突变体。经口投喂后，81-176 recA菌株在兔体内的定殖水平与野生型81-176相当，并且在RITARD（可移除肠系成兔腹泻）模型中能够引发与野生型81-176相同程度的针对后续同源攻击的保护作用。
