生物技术通报
生物技術通報
생물기술통보
BIOTECHNOLOGY BULLETIN
2009年
11期
75-78,82
,共5页
杨尚君%王亚平%唐恩洁%张国元
楊尚君%王亞平%唐恩潔%張國元
양상군%왕아평%당은길%장국원
乙型肝炎病毒%小发夹结构RNA%RNA干扰%乙型肝炎病毒表面抗原
乙型肝炎病毒%小髮夾結構RNA%RNA榦擾%乙型肝炎病毒錶麵抗原
을형간염병독%소발협결구RNA%RNA간우%을형간염병독표면항원
Hepatitis B Virus%shRNA%RNAi%HBsAg
探索用PGenesil-1(Pg)构建的靶向乙型肝炎病毒表面抗原(HBsAg)基因的shRNA表达载体PGenesil-1-HBs(简称Pgs),对体外培养HepG2.2.15细胞中的HBV基因及其抗原表达的抑制作用.设计、合成靶向HBV S区的3对DNA序列,分别插入PGenesil-1中构建3个siRNA表达载体Pgs1、Pgs2、Pgs3,经限制性内切酶,DNA序列测定等技术鉴定确认.筛选并确定最佳细胞接种量及重组质粒转染量后,分别或按不同组合转染HepG-2.2.15细胞,48 h后采用半定量RT-PCR检测HBVsmRNA转录水平,免疫细胞化学技术检测HBsAg表达水平,MEIA分别检测细胞裂解液和培养上清中HBsAg和HBeAg的表达水平.结果表明,HBV真核表达载体Pgs1、Pgs2、Pgs3均能不同程度地抑制HepG2.2.15细胞中的HBsAg、HBeAg合成和HBs-mRNA转录.成功构建的HBV真核表达载体Pgs1、Pgs2、Pgs3,其中PgS3能显著抑制HBsAg表达(P＜0.01).多种表达载体联合对抗原表达的抑制作用效率不同.
探索用PGenesil-1(Pg)構建的靶嚮乙型肝炎病毒錶麵抗原(HBsAg)基因的shRNA錶達載體PGenesil-1-HBs(簡稱Pgs),對體外培養HepG2.2.15細胞中的HBV基因及其抗原錶達的抑製作用.設計、閤成靶嚮HBV S區的3對DNA序列,分彆插入PGenesil-1中構建3箇siRNA錶達載體Pgs1、Pgs2、Pgs3,經限製性內切酶,DNA序列測定等技術鑒定確認.篩選併確定最佳細胞接種量及重組質粒轉染量後,分彆或按不同組閤轉染HepG-2.2.15細胞,48 h後採用半定量RT-PCR檢測HBVsmRNA轉錄水平,免疫細胞化學技術檢測HBsAg錶達水平,MEIA分彆檢測細胞裂解液和培養上清中HBsAg和HBeAg的錶達水平.結果錶明,HBV真覈錶達載體Pgs1、Pgs2、Pgs3均能不同程度地抑製HepG2.2.15細胞中的HBsAg、HBeAg閤成和HBs-mRNA轉錄.成功構建的HBV真覈錶達載體Pgs1、Pgs2、Pgs3,其中PgS3能顯著抑製HBsAg錶達(P＜0.01).多種錶達載體聯閤對抗原錶達的抑製作用效率不同.
탐색용PGenesil-1(Pg)구건적파향을형간염병독표면항원(HBsAg)기인적shRNA표체재체PGenesil-1-HBs(간칭Pgs),대체외배양HepG2.2.15세포중적HBV기인급기항원표체적억제작용.설계、합성파향HBV S구적3대DNA서렬,분별삽입PGenesil-1중구건3개siRNA표체재체Pgs1、Pgs2、Pgs3,경한제성내절매,DNA서렬측정등기술감정학인.사선병학정최가세포접충량급중조질립전염량후,분별혹안불동조합전염HepG-2.2.15세포,48 h후채용반정량RT-PCR검측HBVsmRNA전록수평,면역세포화학기술검측HBsAg표체수평,MEIA분별검측세포렬해액화배양상청중HBsAg화HBeAg적표체수평.결과표명,HBV진핵표체재체Pgs1、Pgs2、Pgs3균능불동정도지억제HepG2.2.15세포중적HBsAg、HBeAg합성화HBs-mRNA전록.성공구건적HBV진핵표체재체Pgs1、Pgs2、Pgs3,기중PgS3능현저억제HBsAg표체(P＜0.01).다충표체재체연합대항원표체적억제작용효솔불동.
It was to construct the shRNA expressing vectors targeting HBsAg gene of HBV, to probe their inhibitation on the HBV antigen expression. Three couple of DNA sequence targeting the HBV S gene was synthesized and cloned into PGenesil-1 plasmid vector,to construct Pgsl ,Pgs2,Pgs3 shRNA expressing vectors,which was confirmed by restriction enzyme digestion,PCR and sequencing. Then, the vectors were respectively or grouping differently transfected into HepG-2. 2. 15 cells. Transcription level of HBs mRNA was tested by means of semiquantitative RT-PCR,and expressional level of HBsAg in HepG-2. 2. 15 cells was tested by ICC. The observed results made sure the Pgsl ,Pgs2,Pgs3 shRNA expression vectors constructed could all inhibit HBsAg expression in the HepG-2. 2.15 in vitro. Thus,the Pgs3 shRNA expression vectors constructed successfully could significantly inhibit HBsAg expression compareed with control group(P <0.01 ) ,while inhibition of combination shRNA expression vector on expression of HBV antigen were not different.
