包装菌株XL-pⅢ的构建及缺陷型辅助噬菌体的制备

Construction of a packaged strain XL-pⅢ and preparation of defective helper phage

目的构建能用来制备基因Ⅲ缺失的辅助噬菌体的包装菌株。方法采用重叠延伸PCR方法将完整的噬菌体基因Ⅲ和氯霉素抗性基因体外拼接起来,利用λRed重组系统将其整合到E.coliXL1-Blue染色体上的组氨酸操纵元位点构建包装菌株,用氯霉素抗性进行筛选,并用PCR及组氨酸表型鉴定;用PCR法构建功能基因Ⅲ缺失的噬菌体基因组,将其转化到包装菌株中培养制备辅助噬菌体,以集落形成来测定滴度。结果重组菌株能在氯霉素抗性平板上生长,PCR扩增可得到目的片段,组氨酸表型缺失,说明靶基因定向重组到了细菌染色体的靶位点,将重组包装菌株命名为XL-pⅢ,以其制备的缺陷噬菌体滴度可达到3.7×108,只具备一次感染力。结论成功构建包装菌株,并能制备辅助噬菌体,有望用于选择感染性噬菌体(SIP)体系。

Objective To construct a packaged bacteria strain and, with it, to prepare gene III-defective helper phage. Methods Whole gene III and chloramphenicol-resistant gene were amplified and joined to form a fragment flanking 36bp homologous sequence of hisBCD using overlapping extending PCR, and it was then integrated into chromosome of E. coli XL1-Blue to replace the hisBCD gene using Red recombination system. The recombinant strain was identified with chloramphenicol-resistant screening, PCR and histidine-phenotype analysis, and named as XL-pⅢ. The gene III-deleted genome of phage was constructed with PCR and transfected into the recombinant strain XL-pⅢ to prepare the defective helper phage. It was quantified by infecting E. coli XL1-Blue and formed colonies were counted in kanamycin plate. Results The recombinant strain could grow in chloramphenicol-resistant plate, but couldn＇t grow in M9 medium without histidine, implying that it had lost its histidine phenotype. The aimed gene fragment could be amplified as designed. The constructed recombinant was named as XL-pⅢ. The prepared defective helper phage could infect E. coli XL1-Blue only once and the CFU was 3.7×108. Conclusions The packaged strain XL-pⅢ is successfully constructed and used to prepare the gene III-deleted helper phage. It is expected to be used in SIP system.