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携带c—myc基因重组腺病毒的构建和鉴定及其在豚鼠耳蜗中的表达与分布
  • ISSN号:1672-4933
  • 期刊名称:《中国听力语言康复科学杂志》
  • 时间:0
  • 分类:R543.5[医药卫生—心血管疾病;医药卫生—临床医学;医药卫生—内科学]
  • 作者机构:[1]第四军医大学西京医院耳鼻咽喉头颈外科,西安710032
  • 相关基金:基金项目:国家自然科学基金(30872857,30930098,30600703)
中文摘要:

目的构建携带c—myc基因的重组腺病毒表达载体,并观察其在豚鼠耳蜗内的表达分布,为探讨该基因的功能奠定实验基础。方法利用细菌内同源重组的方法,构建携带c—myc基因的重组腺病毒表达载体(Ad.c--myc--EGFP),并分别应用酶切、逆转录聚合酶链反应(RT--PCR)和测序方法鉴定腺病毒的构建情况。利用豚鼠耳蜗内显微注射术、Westernblot及荧光显微镜观察注射后该蛋白在耳蜗中的表达及分布特性。结果经酶切、RT--PCR和测序鉴定,质粒构建正确,重组腺病毒Ad.c--myc--EGFP构建成功。Ad.cmmyc--EGFP腺病毒豚鼠耳蜗内注射后第三天便可通过荧光显微镜观察到绿色荧光蛋白的广泛分布,且WesternBlot方法证实它可以上调耳蜗内c-Myc蛋白的表达。结论本实验成功构建了携带c—myc基因的重组腺病毒表达载体,它可以有效地感染豚鼠耳蜗内各种细胞,为深入研究c—myc基因在耳蜗中的作用奠定了实验基础。

英文摘要:

Objective To construct the adenoviral vector carrying c-myc gene and to study the expression of this gene in the cochleae of guinea pigs. Methods The recombinant adenoviral vector carrying the c-myc gene (Ad.c-myc-EGFP) was constructed by homologous recombination in bacteria, and then identified by restriction enzymes and RT-PCR. After Ad. c-myc-EGFP was transferred into the cochleae of guinea pigs, its expression was observed by fluorescence microscopy and detected by Western blot method. Results The recombinant adenoviral vector * carrying c-myc gene was constructed and confirmed by restriction enzyme analysis and RT-PCR. Three days after the inoculation of adenoviral vectors, green fluorescence would be observed in the cochleae, and a single c-Myc band was observed by western blot in the Ad.c-myc-EGFP group. Conclusion The Ad.c-myc-EGFP vector is successfully established and can express efficiently in various cells of cochleae, which lays the foundation for the further research on c-rnyc gene's function in the cochleae.

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期刊信息
  • 《中国听力语言康复科学杂志》
  • 中国科技核心期刊
  • 主管单位:中国残疾人联合会
  • 主办单位:中国聋儿康复研究中心
  • 主编:
  • 地址:北京市朝阳区安外惠新里甲8号
  • 邮编:100029
  • 邮箱:shjournal@163.com
  • 电话:010-84630488 84639344
  • 国际标准刊号:ISSN:1672-4933
  • 国内统一刊号:ISSN:11-5138/R
  • 邮发代号:82-915
  • 获奖情况:
  • 国内外数据库收录:
  • 被引量:2255