目的由华支睾吸虫成虫cDNA文库中筛选重要新基因并进行功能基因研究。方法生物信息学、基因克隆表达技术、免疫识别、S-P免疫组化等方法。结果由华支睾吸虫成虫cDNA文库成功分离到了CsRPEF(RNA聚合酶Ⅱ延长因子)基因。通过BLASTX程序同...目的由华支睾吸虫成虫cDNA文库中筛选重要新基因并进行功能基因研究。方法生物信息学、基因克隆表达技术、免疫识别、S-P免疫组化等方法。结果由华支睾吸虫成虫cDNA文库成功分离到了CsRPEF(RNA聚合酶Ⅱ延长因子)基因。通过BLASTX程序同源性比对,显示CsRPEF基因与小鼠和人类的RPEF基因同源性为82%。成功构建了CsRPEF原核表达载体,并在大肠埃希菌中进行了大规模诱导表达,所表达的GST融合蛋白经蛋白酶原切割后获得CsRPEF重组蛋白。凝胶扫描分析和Bradford法显示,CsRPEF重组蛋白的纯度和浓度分别为85%和(0.73±0.02)mg/ml。将CsRPEF重组蛋白接种至BALB/c小鼠进行免疫实验。间接ELISA法显示免疫小鼠抗体滴度达1∶150。免疫识别结果显示此抗体可识别20 ku CsRPEF重组蛋白和华支睾吸虫20 ku虫源性蛋白。S-P免疫组化方法显示CsRPEF主要定位于华支睾吸虫成虫的表皮、皮下组织和虫卵三部位。CsRPEF新基因序列GenBank的登录号为EU232119。结论CsRPEF基因是防治华支睾吸虫病生物药物研发的重要靶点。展开更多
After reverse transcription, the HIV-1 proviral DNA is integrated into the host genome and thus subjected to transcription by the host RNA polymerase Ⅱ （Pol Ⅱ）. With the identification and characterization of huma...After reverse transcription, the HIV-1 proviral DNA is integrated into the host genome and thus subjected to transcription by the host RNA polymerase Ⅱ （Pol Ⅱ）. With the identification and characterization of human P-TEFb in the late 1990s as a specific host cofactor required for HIV-1 transcription, it is now believed that the elongation stage of Pol Ⅱ transcription plays a particularly important role in regulating HIV-1 gene expression. HIV-1 uses a sophisticated scheme to recruit human P-TEFb and other cofactors to the viral long terminal repeat （LTR） to produce full-length HIV-1 transcripts. In this process, P-TEFb is regulated by the reversible association with various transcription factors/ cofactors to form several multi-subunit complexes （e.g., 7SK snRNP, super elongation complexes （SECs）, and the Brd4-P-TEFb complex） that collectively constitute a P-TEFb network for controlling cellular and HIV-1 transcription. Recent progresses in HIV-1 transcription were reviewed in the paper, with the emphasis on the mechanism and factors that control HIV-1 transcription and latency activation.展开更多
基金Supported by the national high technology re-search and development program of China(863 Program,No.2006AA02Z422)a grant from the key science and technique programof Guangdong Province(No.2004A30801004)
文摘目的由华支睾吸虫成虫cDNA文库中筛选重要新基因并进行功能基因研究。方法生物信息学、基因克隆表达技术、免疫识别、S-P免疫组化等方法。结果由华支睾吸虫成虫cDNA文库成功分离到了CsRPEF(RNA聚合酶Ⅱ延长因子)基因。通过BLASTX程序同源性比对,显示CsRPEF基因与小鼠和人类的RPEF基因同源性为82%。成功构建了CsRPEF原核表达载体,并在大肠埃希菌中进行了大规模诱导表达,所表达的GST融合蛋白经蛋白酶原切割后获得CsRPEF重组蛋白。凝胶扫描分析和Bradford法显示,CsRPEF重组蛋白的纯度和浓度分别为85%和(0.73±0.02)mg/ml。将CsRPEF重组蛋白接种至BALB/c小鼠进行免疫实验。间接ELISA法显示免疫小鼠抗体滴度达1∶150。免疫识别结果显示此抗体可识别20 ku CsRPEF重组蛋白和华支睾吸虫20 ku虫源性蛋白。S-P免疫组化方法显示CsRPEF主要定位于华支睾吸虫成虫的表皮、皮下组织和虫卵三部位。CsRPEF新基因序列GenBank的登录号为EU232119。结论CsRPEF基因是防治华支睾吸虫病生物药物研发的重要靶点。
基金Project supported by the National Natural Science Foundation of China (No.81201276)the Natural Science Foundation of Fujian Province (No.2012J05067),China
文摘After reverse transcription, the HIV-1 proviral DNA is integrated into the host genome and thus subjected to transcription by the host RNA polymerase Ⅱ （Pol Ⅱ）. With the identification and characterization of human P-TEFb in the late 1990s as a specific host cofactor required for HIV-1 transcription, it is now believed that the elongation stage of Pol Ⅱ transcription plays a particularly important role in regulating HIV-1 gene expression. HIV-1 uses a sophisticated scheme to recruit human P-TEFb and other cofactors to the viral long terminal repeat （LTR） to produce full-length HIV-1 transcripts. In this process, P-TEFb is regulated by the reversible association with various transcription factors/ cofactors to form several multi-subunit complexes （e.g., 7SK snRNP, super elongation complexes （SECs）, and the Brd4-P-TEFb complex） that collectively constitute a P-TEFb network for controlling cellular and HIV-1 transcription. Recent progresses in HIV-1 transcription were reviewed in the paper, with the emphasis on the mechanism and factors that control HIV-1 transcription and latency activation.
