Establishment of a Screening System for Selection of siRNA Target Sites Directed against Hepatitis B

来源 :Acta Biochimica et Biophysica Sinica | 被引量 : 0次 | 上传用户:sunnywwh
下载到本地 , 更方便阅读
声明 : 本文档内容版权归属内容提供方 , 如果您对本文有版权争议 , 可与客服联系进行内容授权或下架
论文部分内容阅读
To study the inhibitory effects of plasmid-derived small interfering RNA(siRNA)andsynthetic siRNA on the expression of the hepatitis B virus surface(HBs)gene,three plasmid-derived siRNAsand one synthetic siRNA that complement the coding region of the HBs gene were prepared.The HBsexpression plasmid pHBs-EGFP was also constructed.HeLa cells were co-transfected with pHBs-EGFPand the above siRNAs.The HBs mRNA quantities were measured by reverse-transcription PCR,and thelevel of HBs-EGFP fusion protein was quantified by fluorescent microscope.The concentrations of thehepatitis B virus surface antigen(HBsAg)derived from the culture supernatant of transfected HepG2.2.15cells were measured by an enzyme-linked immunosorbent assay(ELISA)kit.The results showed that thethree plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNAand protein.The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBsexpression.It can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%.To furthersubstantiate the above observations,psiRNA1 was transfected into HepG2.2.15 cells(an HBV secreting cellline).The transfections resulted in almost complete blockage of HBsAg production,whereas control vector-transfected cells secreted high levels of HBsAg 7 days post-transfection.In conclusion,our data suggeststhat RNA interference(RNAi)is an efficient approach for reducing the level of HBs transcripts and proteinsand for suppressing HBsAg production. To study the inhibitory effects of plasmid-derived small interfering RNA (siRNA) andsynthetic siRNA on the expression of the hepatitis B virus surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA that complement the coding region of the HBs gene were prepared The HBs expression plasmid pHBs-EGFP was also constructed. HeLa cells were co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion protein was quantified by fluorescent microscope The concentrations of the hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant of transfected HepG2.2.15 cells were measured by an enzyme-linked immunosorbent assay (ELISA) kit. These results showed that the plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the amount of HBs mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBsexpression. It can inhibit HBs-EG FP expression by 63.3% and suppress HBs mRNA by 75.6% .To furthersubstantiate the above observations, psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cellline). The transfections resulted in almost complete blockage of HBsAg production, but control vector-transfected cells secreted high levels of HBsAg 7 days post-transfection.In conclusion, our data suggest that RNA interference (RNAi) is an efficient approach for reducing the level of HBs transcripts and proteinsand for suppressing HBsAg production.
其他文献
坐落于济南高新技术开发区的成大集团,近几年如一颗新星升起在鲁中大地,自1988年起,已连续8年获省级先进文明单位称号。1994年,经山东省体改委批准,成为济南市第一家符合公
在许多由固相反应生成合金和无机材料的生产过程中,扩散传质控制着反应的过程。但是,对扩散控制固相反应系统的机理和动力学研究还很不成熟。Jander首先把平面扩散抛物线方
通过对《基本科学指标数据库》(ESI)收录的近11年来有关材料学领域的文献进行计量研究,得出材料学领域高生产力和高被引的国家、机构、期刊以及作者的分布,并对材料学领域高
乐山某团3连指导员、市中区茅桥办事处党委书记杨家佑,被誉为预备役部队建设的热心人,连年受到团党委嘉奖,去年被评为“乐山市后备力量建设十佳标兵“之一。 几年来,杨家佑
据近期出版的《自然遗传学》报道,一项新的研究发现一种基因缺陷能够导致机体的两种免疫系统失调,从而使机体有时或常常受到严重的病菌感染。这一发现将促使新的诊断方法诞
由于配位化合物数量庞大,结构特殊,自从它们被合成和发现以来,它们的内部结构一直是化学工作者感兴趣的问题,先后提出了很多理论用来解释形形色色的配位化合物的结构。主要有
[本刊讯]近日,河北省宁晋县中西医结合医院开展了创先争优演讲活动,收到了良好的学习效果。 Recently, Ningjin County, Hebei Province Hospital of Integrated Traditiona
美宝皮康霜是广东省卫生厅1996年初批准汕头美宝制药厂生产的皮肤外用药。为有利于广大医师和皮肤病患者了解美宝皮康霜,精编写《美宝应康霜使用新解》。【科学的组方】美宝皮
目的:观察冷晶体停搏液中加入生理浓度T3对大鼠停搏心肌收缩及舒张功能恢复、心肌酶谱、氧耗量及冠脉内皮细胞功能的影响。方法:运用离体鼠心作功模型,实验组(n=6)含0.6mg/LT3,比较停搏90min前后左室收
加强药检系统精神文明建设的几点建议傅兴治(中国药品生物制品检定所100050)党的十四届六中全会作出的《中共中央关于加强社会主义精神文明建设的若干重要问题的决议》,是新形势下指导