摘要
目的制备地高辛标记的HCV核心区RNA探针,用于筛选与丙型肝炎病毒(HCV)RNA核心区结合的细胞蛋白质分子。方法采用RT-PCR方法,从HCV感染者血清中扩增HCV C区cDNA序列,将所得序列克隆入载体PinpointT-MT Vector和pGEM-7EF(+)Vector,进行PCR、酶切和测序鉴定。以HCV-pGEM-7EF(+)中的HCV核心区cDNA序列作为体外转录RNA的模板,经T7 RNA聚合酶转录得到地高辛标记的HCV核心区RNA探针。将地高辛标记的探针用于紫外交联实验,筛选HepG2细胞中可与HCV RNA结合的蛋白质分子。结果所得HCV核心区cDNA序列为503 bp。地高辛标记的HCV核心区探针初步筛选到两个与HCV RNA结合的蛋白。结论地高辛标记的HCV核心区RNA探针具有较高的特异性和敏感性,可用于筛选与HCV RNA结合的细胞蛋白质分子。
Objective To prepare RNA probe of hepatitis C virus（HCV） core region labeled by digoxingenin and screen cellular proteins binding to HCV RNA from human hepatoma cells, Methods HCV cDNA fragment of core region sequence was amplified by reverse transcription polymerase chain reaction （RT-PCR）. The fragment was cloned into PinpointTM-T Vector and pGEM-7EF （ ＋ ） Vector, then was sequenced and analyzed by restriction endonucleases and PCR. The plasmid HCV-pGEM7zf was used as a template to synthesize the digoxigenin labeled RNA probe with a transcription in vitro. Cellular proteins binding to HCV RNA of core region was screened by ultraviolet （UV） cross-linking with dig-labeled HCV RNA of core region. Results The HCV cDNA sequence of core region was 503 bp. Two cellular proteins of HepG2 were found binding to the core region of HCV RNA labeled by digoxigenin. Conclusions The dig-labeled RNA probe of HCV core region was sensitive and specific, and it may be useful for the study of screening cellular proteins binding to HCV RNA.
出处
《疾病控制杂志》
2006年第4期324-327,共4页
Chinese Journal of Disease Control and Prevention
基金
国家自然科学基金资助面上项目(39970652)
