中华皮肤科杂志 ›› 2004, Vol. 37 ›› Issue (2): 105-108.

• 论著 • 上一篇    下一篇

Ro60 cDNA的克隆及其重组杆状病毒表达载体的构建

郭利劭, 张建中   

  1. 北京大学人民医院皮肤科 100044
  • 收稿日期:2003-03-19 出版日期:2004-02-15 发布日期:2004-02-15
  • 基金资助:
    国家自然科学基金资助(30271194)

Cloning of Human Ro60 cDNA and Construction of Baculovirus Expression Vector

GUO Li-shao, ZHANG Jian-zhong   

  1. Department of Dermatology, Peking University People's Hospital, Beijing 100044, China
  • Received:2003-03-19 Online:2004-02-15 Published:2004-02-15

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摘要： 目的 构建人Ro60cDNA的杆状病毒表达载体。方法 采用逆转录-PCR技术从Hela细胞RNA中扩增Ro60cDNA,定向插入杆状病毒转移载体pFastBacHTc,转化大肠杆菌DH10Bac进行转座,提取重组Bacmid,通过蓝白斑筛选和PCR进行鉴定。结果 成功从Hela细胞RNA中扩增出1.56kbRo60,所克隆的Ro60cDNA与已公布的序列完全符合,证明本克隆为Ro60cDNA的精确拷贝,并证实其目的基因与重组载体发生了特异转座和病毒重组,成功地构建了重组杆状病毒表达载体Bacmid-Ro60。结论 本实验成功克隆了Ro60cDNA并构建了其杆状病毒表达载体,为进一步表达Ro60蛋白和研究此抗原在红斑狼疮的发病机制奠定了基础。

关键词: 基因复制, 遗传载体, Ro60

Abstract: Objective To construct a baculovirus expression vector for expression of human Ro60 cDNA. Methods Ro60 cDNA was cloned from Hela cell RNA with RT-PCR and was inserted into plasmid pFastBac HTc. Plasmid pFastBac HTc-Ro60 was then transformed into E. coli DH10Bac competent cells for transposition. The constructed recombinant was determined by both blue-white colony screening and PCR analysis. Results A 1.56kb Ro60 cDNA was obtained from Hela cell RNA and was successfully inserted to plasmid pFastBac HTc. DNA sequencing and restriction enzyme analysis revealed that both open reading frame and sequence of the cDNA were identical to Ro60 sequence previously published. Specific transposition and virus recombination were obtained in the vector Bacmid-Ro60. Conclusion Ro60 cDNA has been successfully cloned and constructed to baculovirus expression vector, which is useful for protein expression and further study of this autoantigen in the pathogenesis of lupus erythematosus.

Key words: Gene duplication, Genetic vectors, Ro60