用于制备基因Ⅲ缺失的辅助噬菌体的包装菌株的构建

Construction of Packaging Bacterial Strain for Preparation of Gene Ⅲ-deleted Helper Phage

目的利用λ噬菌体Red重组酶系统,构建用于制备基因Ⅲ缺失的辅助噬菌体的包装菌株。方法采用PCR方法,将完整的噬菌体基因Ⅲ和氯霉素抗性基因拼接,使其两端带有与四环素基因同源的36nt序列;利用λ噬菌体Red重组酶系统,将其整合到大肠杆菌XL1-Blue的附加体上,以替换掉四环素基因,经氯霉素抗性筛选及PCR鉴定,获得包装菌株XLe-pⅢ;将基因Ⅲ缺失的噬菌体基因组导入包装菌株,制备缺陷型辅助噬菌体,集落形成试验检测缺陷噬菌体的感染能力。结果所构建的包装菌株XLe-pⅢ经氯霉素抗性筛选及PCR鉴定,证明氯霉素抗性基因已与噬菌体基因Ⅲ一同整合到附加体上,重组菌不能在含有氨苄西林的LB培养基中生长,仍保留四环素抗性;该包装菌株能用于制备基因Ⅲ缺失的辅助噬菌体,制备的缺陷型辅助噬菌体的滴度为3.0×108。结论利用λ噬菌体Red重组酶系统,已成功构建用于制备基因Ⅲ缺失的辅助噬菌体的包装菌株,以其制备的缺陷型辅助噬菌体有望用于常规筛选或SIP体系。

Objective To construct a packaging bacterial strain for preparation of gene Ⅲ-deleted helper phage. Methods The full-length gene Ⅲ of phage and chloramphenicol-resistant gene were spliced by PCR to form a gene fragment flanked with 36 nt sequence homologous to tetracycline （Tet） gene. The PCR product was integrated to the episome of E. coli XL1-Blue by using Red recombination enzyme system of λ phage to substitute for Tet gene. The packaging strain Xle-p Ⅲ was screened by chloramphenicolresistant screening and identified by PCR. Defective phage was prepared by transforming gene Ⅲ-deleted phage genome to the packaging strain and determined for infective ability by colony formation test. Results The chloramphenicol-resistant screening and PCR identification of constructed packing strain Xle-p Ⅲ proved that both chloramphenicol-resistant gene and phage gene Ⅲ were integrated to the episome of E. coli XL1-Blue. The recombinant strain could not grow in the LB medium containing ampicillin but remained Tet-resistance. The titer of defective phage prepared with the packaging strain was 3.0 × 10^8. Conclusion The packaging bacterial strain for preparation of gene Ⅲ-deleted helper phage was successfully constructed by using λRed recombination enzyme system, and the prepared defective helper phage might be used for routine screening or SIP system.