中国医科大学学报

中国医科大学学报
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中国医科大学学报 ›› 2018, Vol. 47 ›› Issue (2): 102-106.doi: 10.12007/j.issn.0258-4646.2018.02.002

• 论著 • 上一篇    下一篇

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

柳中洋1, 卢颖2, 韩丽英3, 马艳萍1, 齐莹1, 黄郁晶1, 阮强1   

  1. 1. 中国医科大学附属盛京医院病毒研究室, 沈阳 110004;
    2. 锦州医科大学基础医学院病原生物学教研室, 辽宁 锦州 121001;
    3. 上海交通大学附属儿童医院新生儿科, 上海 200062
  • 收稿日期:2017-07-14 出版日期:2018-02-28 发布日期:2018-01-11
  • 通讯作者: 阮强 E-mail:ruanq@sj-hospital.org
  • 作者简介:柳中洋(1985-),男,研究实习员,硕士.
  • 基金资助:
    国家自然科学基金(81371788)

Application of Bacterial Artificial Chromosome in Small Fragment Mutation of the Human Cytomegalovirus Genome

LIU Zhongyang1, LU Ying2, HAN Liying3, MA Yanping1, QI Ying1, HUANG Yujing1, RUAN Qiang1   

  1. 1. Virus Laboratory, Shengjing Hospital, China Medical University, Shenyang 110004, China;
    2. Department of Immunology and Pathogenic Biology, School of Basic Medicine, Jinzhou Medical University, Jinzhou 121001, China;
    3. Neonatal Department, Children's Hospital of Shanghai Jiaotong University, Shanghai 200062, China
  • Received:2017-07-14 Online:2018-02-28 Published:2018-01-11

摘要: 目的 评估细菌人工染色体技术在人巨细胞病毒(HCMV)基因组小片段序列突变中的作用。方法 选取HCMV LUNA基因位于UL80UL82 2个基因之间的31 bp片段作为突变靶点,用含有同源臂的卡那霉素抗性序列对目的片段进行同源重组置换,电转化法对突变成功的病毒进行拯救,反转录PCR和cDNA克隆测序对LUNAUL80UL82 mRNA转录情况和转录本3'末端结构进行分析。结果 目的基因片段缺失成功,获得的单克隆中突变成功的比例>3%,LUNA转录起始端序列突变的病毒拯救成功,UL80UL82的mRNA转录与转录本3'结构未受影响。结论 通过人工染色体技术成功进行了HCMV Han株LUNA基因转录起始端序列突变,HCMV Han株细菌人工染色体支持有效缺失小至31 bp的基因片段。

关键词: 人巨细胞病毒, 细菌人工染色体, LUNA, 突变

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.

Key words: cytomegalovirus, bacterial artificial chromosome, LUNA, mutation

中图分类号: 

  • R373.9
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