Application of Bacterial Artificial Chromosome in Small Fragment Mutation of the Human Cytomegalovirus Genome / 中国医科大学学报

ABSTRACT

Objective To evaluate the applicability of Han strain bacterial artificial chromosome (Han-BAC) in small fragment mutation of the human cytomegalovirus (HCMV) genome. Methods A 31-bp long fragment of LUNA between UL80 and UL82 in the HCMV was chosen as the mutation target. Kanamycin resistance gene sequence flanking the homologous arms of the target neighbor sequence was used to replace the target sequence. Electronic transformation was used to rescue the mutated virus. Reverse transcription PCR and cDNA clone sequencing were used to identify the mRNA expression and the 3' terminal structures of LUNA,UL80,and UL82 transcripts. Results The 31 bp fragment was replaced precisely by the kanamycin resistance gene sequence. The efficiency of mutation was more than 3％. LUNA-mutated virus was rescued successfully. Transcriptions and 3' terminal structures of the UL80 and UL82 transcripts of the mutant virus were the same as those of its original virus. Conclusion The sequence at the transcription start site of LUNA was replaced successfully. The HCMV Han-BAC supports fragment mutation as small as 31 bp with a relatively high efficiency.