摘要
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.
出处
《中国生物制品学杂志》
CAS
CSCD
2008年第9期746-748,共3页
Chinese Journal of Biologicals
基金
国家自然科学基金资助项目(30200156)