中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (2): 311-314.doi: 10.3969/j.issn.1673-8225.2012.02.028

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

人端粒酶催化亚基反义RNA重组腺病毒载体的构建与鉴定*

彭  智，贾振华，唐红伟，黄海华，郭晓瑞，韦有万，王香梅，张培华   

  1. 广东医学院附属医院整形外科，广东省湛江市  524001
  • 收稿日期:2011-03-19 修回日期:2011-03-25 出版日期:2012-01-08 发布日期:2012-01-08
  • 作者简介:Peng Zhi, Chief physician, Master’s supervisor, Department of Plastic Surgery, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, Guangdong Province, China pengzhibb@163.com
  • 基金资助:

    广东省社会发展项目 (2007B031510001)，hTERT调控区反义RNA腺病毒载体的构建及其对瘢痕疙瘩成纤维细胞作用。

Construction and identification of recombinant adenovirus vector of human telomerase reverse transcriptase

Peng Zhi, Jia Zhen-hua, Tang Hong-wei, Huang Hai-hua, Guo Xiao-rui, Wei You-wan, Wang Xiang-mei, Zhang Pei-hua   

  1. Department of Plastic Surgery, Affiliated Hospital of Guangdong Medical University, Zhanjiang   524001, Guangdong Province, China
  • Received:2011-03-19 Revised:2011-03-25 Online:2012-01-08 Published:2012-01-08
  • About author:彭智，男，1966年生，广东省湛江市人，汉族，1989年广东医学院毕业，主任医师，硕士生导师，主要从事病理性瘢痕的基础及临床研究和脂肪干细胞的基础及临床研究。pengzhibb@163.com
  • Supported by:

     the Social Development Program of Guangdong Province, No. 2007B031510001*

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摘要：

背景：人端粒酶催化亚基与c-myc在喉鳞状细胞癌中有密切的相关性。
目的：构建含有c-myc启动子的人端粒酶催化亚基的重组腺病毒载体，观察人端粒酶催化亚基在细胞中表达后对细胞增长的影响。
方法：按照人工合成中反向转录的方法设计基因合成引物，用复式PCR获得目的片段ashTERT(322 bp)。将片段ashTERT克隆到pUC57(2.7 kb)克隆载体，然后热激转化到E.coli感受态细胞中，菌检、PCR、鉴定阳性克隆后，菌液放大培养，提取质粒pUC57-ashTERT，然后对质粒进行测序。将目的基因片段亚克隆到穿梭载体pshuttle-CMVneo上，然后构建重组腺病毒质粒pAd-ashTERT。转入293细胞中进行病毒包装、鉴定和滴度测定。
结果与结论：提取质粒pUC57-ashTERT，测序符合序列要求。质粒pUC57-ashTERT转入293细胞后，成功构建了有较强感染能力的含人端粒酶催化亚基基因的重组腺病毒载体。

关键词: 人端粒酶催化亚基, 瘢痕疙瘩, 成纤维细胞, 腺病毒, 基因治疗

Abstract:

BACKGROUND: Human telomerase reverse transcriptase (hTERT) closely correlates with c-myc in laryngeal squamous cell carcinoma.
OBJECTIVE: To construct recombinant adenovirus vector of hTERT containing c-myc promoter and to observe the effects of expressed hTERT on cell growth.
METHODS: Using the method of reverse transcription in artificial synthesis to design the gene synthesis primer and target fragment ashTERT (322 bp) was obtained using overlapping PCR. The target fragment ashTERT was cloned into the vector pUC57(2.7 kb) and then heat-shock transformed into the competent cells E.coli. With Bacteria inspection, PCR, identification of positive clones, plasmid pUC57-ashTERT was extracted and sequenced. The target gene fragments were subcloned into the shuttle vector pshuttle-CMVneo and then the recombinant adenovirus plasmid pAd-ashTERT was constructed. Subsequently, the recombinant adenovirus plasmid pAd-ashTERT was transformed into the 293A cells for package, identification and titer determination of adenovirus.
RESULTS AND CONCLUSION: pUC57-ashTERT plasmid was extracted and its sequence corresponded to sequencing requirement. Recombinant adenovirus vector of hTERT with relatively strong ability of infection was successfully constructed after pUC57-ashTERT plasmid was transformed into 293 cells.

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