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构建痢疾弗氏志贺菌 2aT32的asd基因全缺失突变体。方法 :采用全菌PCR技术 ,从痢疾弗氏 2aT32株染色体扩增了asd基因及其上下游各约 50 0bp的染色体DNA序列 ,并将其克隆至 pUC18,测定其核苷酸序列 ,在体外用ctxB基因置换了asd基因 ,然后将其克隆至自杀载体pXL2 75。通过细菌交配和体内同源重组 ,用ctxB基因完全取代痢疾弗氏 2aT32株染色体上的asd基因。结果与结论 :构建成asd基因全缺失而稳定表达CtxB的T32突变株FWL0 1,为进一步用载体 /宿主平衡致死系统构建多价菌苗候选株打下了基础。
Construction of asd gene deletion mutant of Shigella dysenteriae 2aT32. Methods: Asd gene and its deduced 50 bp upstream and downstream chromosomal DNA sequences were amplified from the genome of S.Taii 2aT32 strain by whole-bacteria PCR and cloned into pUC18. The nucleotide sequence was determined in vitro The ctxB gene replaces the asd gene, which is then cloned into the suicide vector pXL275. The ctxB gene completely replaced the asd gene on the chromosome of chromosome 2aT32 in dysentery by bacterial mating and in vivo homologous recombination. RESULTS AND CONCLUSION: The constructed T32 mutant strain FWL0 1 with full deletion of asd gene and stable expression of CtxB laid the foundation for further construction of candidate strains of multivalent vaccine strains by the carrier / host balance lethal system.