Application of Bacterial Artificial Chromosome in Small Fragment Mutation of the Human Cytomegalovirus Genome
10.12007/j.issn.02584646.2018.02.002
- VernacularTitle:细菌人工染色体技术在人巨细胞病毒基因组小片段序列置换突变中的应用
- Author:
Zhongyang LIU
1
;
Ying LU
;
Liying HAN
;
Yanping MA
;
Ying QI
;
Yujing HUANG
;
Qiang RUAN
Author Information
1. 中国医科大学附属盛京医院病毒研究室
- Keywords:
cytomegalovirus;
bacterial artificial chromosome;
LUNA;
mutation
- From:Journal of China Medical University
2018;47(2):102-106
- CountryChina
- Language:Chinese
-
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.
