摘要
目的:构建带有琥珀突变的辅助病毒VCSM13N1,为建立选择感染性噬菌体(SIP)技术平台奠定基础。方法:选择辅助病毒VCSM13蛋白ⅢN1区的Q51作为靶位点,采用重叠延伸PCR方法对其进行定点突变,经酶切及序列分析进行确认;噬斑计数法检测该琥珀突变在允许和非允许菌株中的开、关效应;以HBsAg抗体作为模式体系来确定该突变子作为辅助病毒的可行性,并对其性能进行综合评价。结果:获得了琥珀突变子VCSM13N1,在允许菌株XL1-Blue中其滴度达到2.5×1011,与野生型VCSM13相似(4×1011),而用非许可菌株HB2151进行噬斑计数时,滴度在106水平,降低了数万倍。来源于菌株HB2151的突变病毒感染XL1-Blue计数噬斑,其滴度最高为5×109,降低了50倍;以其在许可菌中制备HBsAg噬菌体抗体,可以得到与野生型相似的结果。结论:琥珀突变子VCSM13N1可以作为辅助病毒,而且在非允许菌株HB2151中有一定的关闭效应。
Objective：To construct the helper virus VCSM13N1 with amber mutation,so as to provide a platform for the selective infective phage（SIP） technique. Methods： A pair of primers with amber stop codon were synthesized and introduced into N1 region of helper virus VCSM13 protein by overlapping extension PCR, and the products were identified by ristriction enzyme digestion and sequencing. The switch on/off efficiency of amber codon was detected with E. coli XL1-Blue and HB2151. The feasibility of using the amber mutant phage as helper virus was determined with anti-HBsAg phage antibody. Comprehensive analysis was also made on the performance of the amber mutant phage. Results.. The titer of mutant phage was 2.5× 10^11 when detected with E. coli XL1-Blue,comparable with that of wild phage. The titer of mutant phage was at 106 level when E. coli HB2151 was used, being 10 000 folds lower than that of wild phage, when the mutant phages from HB2151 were used to infect E. coli XL1-Blue, the titer peaked at 5×10^9 , being 50 folds lower than that of wild phage. The activity of phage antibody prepared with mutant virus was comparable with the phage antibody prepared with wild phage. Conclusion： The constructed amber mutant phage can be used as helper virus,and it can switch off efficiently in non-suppressing strain HB2151.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2005年第12期1340-1342,共3页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(30200156)
关键词
突变
辅助病毒
选择感染性噬菌体
mutation
helper viruses
selectively infective phage
