Construction of helper virus VCSM13N1 with amber mutation / 第二军医大学学报

ABSTRACT

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 10~6 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.