中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2010年
39期
2776-2781
,共6页
边中启%孙璐璐%陈维灶%肖安%马世武%崔志磊%刘霜%刘明秋%严维耀%郑兆鑫
邊中啟%孫璐璐%陳維竈%肖安%馬世武%崔誌磊%劉霜%劉明鞦%嚴維耀%鄭兆鑫
변중계%손로로%진유조%초안%마세무%최지뢰%류상%류명추%엄유요%정조흠
肝炎病毒,乙型%RNA干扰%短发夹状RNA%微RNA%应急疫苗
肝炎病毒,乙型%RNA榦擾%短髮夾狀RNA%微RNA%應急疫苗
간염병독,을형%RNA간우%단발협상RNA%미RNA%응급역묘
Hepatitis B virus%RNAi%shRNA%miRNA%Emergency vaccine
目的 研究靶向乙型肝炎病毒(HBV)C基因的shRNA(shRNA)诱导RNAi(RNAi)在BHK-21细胞中抑制HBV的复制与表达及抗病毒效果.方法 根据成都军区昆明总医院传染病中心克隆测序的中国56个民族CHB患者HBV基因组(基因型B属ayw1亚型)已在GenBank登录注册:CYN/2002和CYN/2000(GenBank登录号AY517488,AY517489,等),为了监测siRNA功能提供报告基因系统,将HBV C基因PCR产物克隆构建成报告基因表达载体pC-EGFP-N1和载体pCDNA3.1B(-),设计并构建两个靶向同源(CYN/2002和CYN/2000)毒株HBV C基因的长24核苷酸(nt)的shRNA表达质粒(S1和S2),随机设计的用于对照的非同源长24 nt的shRNA表达质粒S3,将其克隆到载体pU6上,构建成表达目的 shRNA重组表达载体,并与pC-EGFP-N1共转染BHK-21细胞.首先在BHK-21细胞中使用BH-2型荧光显微镜观察和FACS-440型流式细胞仪检测绿色荧光蛋白(EGFP)表达细胞数量,评估该shRNA表达质粒在转染后不同时间对C基因/EGFP融合报告基因表达的抑制作用,接着在BHK-21细胞中通过实时荧光定量PCR(RT-PCR)进一步检验了shRNA抗病毒效果.结果 通过BH-2型荧光显微镜观察和FACS-440型流式细胞仪检测,发现在shRNA表达质粒和pC-EGFP-N1共转染BHK-21细胞24 h后,与pC-EGFP-N1或pEGFP-N1单质粒转染相比,S1或S2的共转染组、S1+S2的共转染组使EGFP的表达水平降低了90%,而对照质粒S3或pU6共转染组无显著降低EGFP的表达水平,差异有统计学意义(P＜0.01);应用RT-PCR检测C基因mRNA和EGFP基因mRNA的表达量,结果与BH-2型荧光显微镜和FACS-440型流式细胞仪检测结果相吻合,差异有统计学意义(P＜0.01).进一步证实了RNAi抗病毒效果,抗病毒效应持续时间超过48 h.结果 发现,创建的靶向C基因的shRNA能够有效特异地抗C-EGFP基因在BHK-21细胞中的复制与表达.结论 靶向C基因的RNAi技术能够有效特异地抗HBV在BHK-21细胞中的复制与表达.RNAi可能成为抗重大传染病HBV/HCV安全有效的应急疫苗.
目的 研究靶嚮乙型肝炎病毒(HBV)C基因的shRNA(shRNA)誘導RNAi(RNAi)在BHK-21細胞中抑製HBV的複製與錶達及抗病毒效果.方法 根據成都軍區昆明總醫院傳染病中心剋隆測序的中國56箇民族CHB患者HBV基因組(基因型B屬ayw1亞型)已在GenBank登錄註冊:CYN/2002和CYN/2000(GenBank登錄號AY517488,AY517489,等),為瞭鑑測siRNA功能提供報告基因繫統,將HBV C基因PCR產物剋隆構建成報告基因錶達載體pC-EGFP-N1和載體pCDNA3.1B(-),設計併構建兩箇靶嚮同源(CYN/2002和CYN/2000)毒株HBV C基因的長24覈苷痠(nt)的shRNA錶達質粒(S1和S2),隨機設計的用于對照的非同源長24 nt的shRNA錶達質粒S3,將其剋隆到載體pU6上,構建成錶達目的 shRNA重組錶達載體,併與pC-EGFP-N1共轉染BHK-21細胞.首先在BHK-21細胞中使用BH-2型熒光顯微鏡觀察和FACS-440型流式細胞儀檢測綠色熒光蛋白(EGFP)錶達細胞數量,評估該shRNA錶達質粒在轉染後不同時間對C基因/EGFP融閤報告基因錶達的抑製作用,接著在BHK-21細胞中通過實時熒光定量PCR(RT-PCR)進一步檢驗瞭shRNA抗病毒效果.結果 通過BH-2型熒光顯微鏡觀察和FACS-440型流式細胞儀檢測,髮現在shRNA錶達質粒和pC-EGFP-N1共轉染BHK-21細胞24 h後,與pC-EGFP-N1或pEGFP-N1單質粒轉染相比,S1或S2的共轉染組、S1+S2的共轉染組使EGFP的錶達水平降低瞭90%,而對照質粒S3或pU6共轉染組無顯著降低EGFP的錶達水平,差異有統計學意義(P＜0.01);應用RT-PCR檢測C基因mRNA和EGFP基因mRNA的錶達量,結果與BH-2型熒光顯微鏡和FACS-440型流式細胞儀檢測結果相吻閤,差異有統計學意義(P＜0.01).進一步證實瞭RNAi抗病毒效果,抗病毒效應持續時間超過48 h.結果 髮現,創建的靶嚮C基因的shRNA能夠有效特異地抗C-EGFP基因在BHK-21細胞中的複製與錶達.結論 靶嚮C基因的RNAi技術能夠有效特異地抗HBV在BHK-21細胞中的複製與錶達.RNAi可能成為抗重大傳染病HBV/HCV安全有效的應急疫苗.
목적 연구파향을형간염병독(HBV)C기인적shRNA(shRNA)유도RNAi(RNAi)재BHK-21세포중억제HBV적복제여표체급항병독효과.방법 근거성도군구곤명총의원전염병중심극륭측서적중국56개민족CHB환자HBV기인조(기인형B속ayw1아형)이재GenBank등록주책:CYN/2002화CYN/2000(GenBank등록호AY517488,AY517489,등),위료감측siRNA공능제공보고기인계통,장HBV C기인PCR산물극륭구건성보고기인표체재체pC-EGFP-N1화재체pCDNA3.1B(-),설계병구건량개파향동원(CYN/2002화CYN/2000)독주HBV C기인적장24핵감산(nt)적shRNA표체질립(S1화S2),수궤설계적용우대조적비동원장24 nt적shRNA표체질립S3,장기극륭도재체pU6상,구건성표체목적 shRNA중조표체재체,병여pC-EGFP-N1공전염BHK-21세포.수선재BHK-21세포중사용BH-2형형광현미경관찰화FACS-440형류식세포의검측록색형광단백(EGFP)표체세포수량,평고해shRNA표체질립재전염후불동시간대C기인/EGFP융합보고기인표체적억제작용,접착재BHK-21세포중통과실시형광정량PCR(RT-PCR)진일보검험료shRNA항병독효과.결과 통과BH-2형형광현미경관찰화FACS-440형류식세포의검측,발현재shRNA표체질립화pC-EGFP-N1공전염BHK-21세포24 h후,여pC-EGFP-N1혹pEGFP-N1단질립전염상비,S1혹S2적공전염조、S1+S2적공전염조사EGFP적표체수평강저료90%,이대조질립S3혹pU6공전염조무현저강저EGFP적표체수평,차이유통계학의의(P＜0.01);응용RT-PCR검측C기인mRNA화EGFP기인mRNA적표체량,결과여BH-2형형광현미경화FACS-440형류식세포의검측결과상문합,차이유통계학의의(P＜0.01).진일보증실료RNAi항병독효과,항병독효응지속시간초과48 h.결과 발현,창건적파향C기인적shRNA능구유효특이지항C-EGFP기인재BHK-21세포중적복제여표체.결론 파향C기인적RNAi기술능구유효특이지항HBV재BHK-21세포중적복제여표체.RNAi가능성위항중대전염병HBV/HCV안전유효적응급역묘.
Objective The vaccines currently developed against infectious diseases fail to induce effective antiviral immune responses to control an abrupt outbreak of viral diseases epidemic in a short space of time.Hence there is an urgent need to develop emergency vaccines capable of producing prophylactic effects against infectious diseases.RNA interference(RNAi)is a mechanism that inhibits gene expression by causing the degradation of specific RNA molecules or hindering the transcription of specific genes.The rapidity and uniqueness of RNAi responses can make up for the current inadequacy of antiviral preventive regimes.Here we evaluate the antiviral potential of short hairpin RNA(shRNA)targeting C(core)gene of hepatitis B virus(HBV).It plays essential roles in HBcAg encoding and viral attachment to susceptible cells during its life cycle.The present study was intended to investigate the inhibitory effect of C-specific shRNAs on HBV replication and expression in BHK-21 cells.Methods The reporter gene expression vector of pC-EGFP-N1 was constructed by cloning the DNA(PCR product)of HBV C into the EcoR Ⅰ-Hind Ⅲ sites of pEGFP-N1 to fuse C to enhanced green fluorescent protein(EGFP)for providing a reporting system for monitoring siRNA function.Plasmid pC was constructed by cloning the DNA of HBV C into the EcoR Ⅰ-Hind Ⅲ sites of pCDNA3.1B(-)directly under the control of cytomegalovirus promoter.Two plasmids(S1 & S2)were constructed to express shRNAs targeting C of HBV with the length of 24 nucleotide(nt)homologous in sequence to the HBV C gene.Plasmids were designed and synthesized according to the HBV genome(HBV genotype B, ayw1 subtype)of chronic hepatic B patients from 56 ethnic minorities in China.After cloning and sequencing, the investigators registered them with the GenBank accession numbers of AY517488(CYN/2002)and AY517489(CYN/2000), etc.Simultaneously, one of nonspecific shRNA-S3 with a length of 24 nt was also designed randomly for negative control.After cloning into vector pU6 for constructing shRNA-expressing plasmids, they were then cotransfected into BHK-21 cells along with reporter gene expression vector of pC-EGFP-N1.First, upon a determination of the number of cells exhibiting EGFP expression in BHK-21 cells as detected by an Olympus BH-2 fluorescence microscope and FACS-440 flow cytometry(Becton-Dickinson, USA)at different times after cotransfection, the investigators evaluated the gene inhibitory efficiency of both plasmids by an EGFP reporter system in BHK-21 cells.Subsequently, the antiviral efficacy of both plasmids in BHK-21 cells was further investigated by real-time quantitative polymerase chain reaction.Results In comparison with single plasmid transfection pC-EGFP-N1 or pEGFP-N1, fluorescence microscope and flow cytometry detection at 24 h post-cotransfection revealed that the expression of reporter gene EGFP in cotransfection group involving S1 or S2 and S1 + S2 cotransfection group was reduced significantly by about 90% in EGFP signal versus the control.And the EGFP expression in control plasmid cotransfected S3 or pU6 was not significantly reduced in BHK-21 cells(P ＜ 0.01).It was found that the expression of mRNAs of HBV C and EGFP gene as detected by real-time quantitative PCR was the same as that detected by fluorescence microscope and flow cytometry(P ＜ 0.01), thereby further corroborating the antiviral efficacy of RNAi.The antiviral efficacy extended to almost 48 hours.The results indicted that a DNA vector-based RNAi technology could effectively and specifically inhibit the replication and expression of HBV in BHK-21 cells.Conclusion For the first time it has been found that RNAi induced by shRNA targeting C gene exerts an effective and unique inhibition of HBV replication and expression in BHK-21 cells.Thus RNAi may provide an effective emergency vaccine against major infectious diseases such as HBV infection.
