重组质粒载体和禽腺联病毒介导的miRNA抑制IBDV复制研究
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摘要
传染性法氏囊病(Infectious bursal disease,IBD)是由传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)引起的一种鸡的高度接触性传染病,除引起鸡死亡和生产性能下降,还可导致感染鸡的免疫抑制和其它疫苗接种的免疫失败。IBD呈世界范围流行,尤其是近年来超强毒株和抗原变异株的出现,使得常规疫苗的免疫效果明显下降,给养鸡业造成的危害越来越大,迫切需要更为有效的新型防制措施。
RNA干扰(RNA interference,RNAi)是近年来快速发展的一种转录后基因沉默技术,在功能基因组研究、肿瘤治疗和抗病毒感染等方面具有良好的应用前景,已被证明能抑制几乎所有种属病毒的复制。为探索用RNAi技术抑制IBDV复制的可行性,本研究在比较不同来源RNA聚合酶启动子转录活性基础上,选用禽源U6启动子构建短干扰RNA(short interference RNA,siRNA)表达载体,以绿色荧光蛋白(green fluorescent protein,GFP)基因为报告基因,证明在禽源细胞中能成功实现特异基因表达沉默;进而用专业软件预测IBDV VP2基因特异性siRNA,将人工合成的微小RNA(microRNA,miRNA)插入表达载体,以细胞转染法筛选出2个有效抑制IBDV VP2基因表达的miRNA,将其克隆入含neo基因的表达载体,经G418筛选获得抗性细胞克隆,用感染试验证明能显著抑制IBDV复制;最后将miRNA表达盒插入禽腺联病毒(avian adeno-associated virus,AAAV)转移载体,用重组病毒感染细胞进行的试验结果证明,表达的miRNA对IBDV复制不仅具有更为显著的抑制作用,而且对异源毒株复制也有类似的抑制效果,为IBD防制研究开辟了新的途径,现将试验结果总结如下。
目前siRNA表达试验多用人或鼠源H1或U6启动子在哺乳动物细胞中进行,对于这些启动子能否在禽细胞中有效转录短发夹RNA(small hairpin RNA,shRNA)或miRNA仍有争议。我们利用siDirect软件预测GFP基因特异性siRNA,将人工合成的相应shRNA插入含人H1启动子的pSuper载体,获得重组载体pSuper-shRNA;再将shRNA表达盒插入含GFP基因的pEGFP-N1载体,获得重组载体pGFP-H1-shRNA。分别以pEGFP-N1、pSuper-shRNA+pEGFP-N1和pGFP-H1-shRNA转染哺乳动物源COS-1、293-T细胞和禽源鸡胚肝(CEL)、鸡胚成纤维(CEF)细胞,根据相同条件下转染细胞培养中荧光阳性细胞数及荧光强度的变化,比较人H1启动子在哺乳动物源和禽源细胞中转录shRNA的效率。结果显示,人H1启动子在哺乳动物细胞中能有效转录shRNA,但在禽源细胞中的转录效率很低。这些试验结果表明,开展禽源细胞siRNA表达研究应选用禽源启动子。
为了开展禽源细胞RNAi研究,以含鸡U6启动子的pRFPRNAiC为miRNA表达载体,用Genscript软件预测GFP序列特异性siRNA,从10个潜在的siRNA中随机选择1个siRNA序列,用PCR合成双链寡核苷酸,将其插入表达载体pRFPRNAiC,用获得的重组载体pRFPRNAiGFP与报告质粒pEGFP-N1共转染COS-1、293-T、CEL和CEF细胞,根据相同条件下GFP阳性细胞数及荧光强度变化,比较哺乳动物和禽细胞中鸡U6启动子表达miRNA的效率。结果显示,pRFPRNAiGFP在哺乳动物和禽源细胞中都能表达抑制性miRNA,在禽源细胞中转录效率较高。这些试验结果表明,源禽U6启动子转录miRNA的细胞种属特异性相对较弱,pRFPRNAiC载体可用于禽源细胞的miRNA表达研究。
为了探索用RNAi技术抑制IBDV复制的可行性,利用Genscript软件预测针对IBDV VP2基因的siRNA,从10个潜在的siRNA中选择5个siRNA序列,将PCR产生的双链寡核苷酸插入pRFPRNAiC载体,获得重组载体pRFPRNAmiVP2A、pRFPRNAmiVP2B、pRFPRNAmiVP2C、pRFPRNAmiVP2D、pRFPRNAmiVP2E;根据IBDV Lukert株VP2基因序列设计引物,用PCR扩增不含自身翻译终止码的VP2基因,将扩增产物与pEGFP-N1载体中GFP基因的N端融合,获得报告载体pVP2-GFP。分别将5个miRNA表达载体与报告载体共转染DF-1细胞,在转染后24h用Northern Dot Blotting确认miRNA表达,然后分别用荧光共聚焦显微镜和流式细胞术对转染细胞培养中的荧光阳性细胞数及荧光总量进行定性和定量分析。结果与报告载体单独转染细胞相比,5个miRNA表达载体与报告载体共转染细胞的荧光抑制效率在59.7%-78.5%之间;将抑制效果较好的miVP2A和miVP2E表达盒分别克隆入含neo基因的pTarget载体,用获得的重组载体转染DF-1细胞,经G418筛选后获得抗性细胞,用IBDV Lurket感染,感染三天后用半定量RT-PCR测定VP2基因表达,并测定IBDV半数组织细胞感染剂量(TCID_(50))。结果显示,miVP2A和miVP2E表达细胞中VP2基因表达抑制率分别为80.7%和75.0%,TCID_(50)分别下降6和5lgs。这些试验结果表明,针对VP2基因的两个miRNA对IBDV基因表达和复制均有显著的抑制作用。
利用重组AAAV稳定表达外源基因的特点,用限制酶消化法去除含AAAV全基因组质粒载体pCR-AAAV中的Pep和Cap蛋白编码序列,获得AAAV转移载体pAITR;分别将miVP2E和针对VP1基因的miVP1表达盒连同红色荧光蛋白(redfluorescent protein,RFP)基因表达盒插入pAITR中左、右ITR之间,分别用获得的重组载体pAITR-RFP-miVP2E和pAITR-RFP-miVP1与AAAV包装载体pcDNA-ARC及腺病毒辅助载体pHelper共转染AAV-293细胞,经PCR检测证明获得的rAAAV中含有miRNA表达盒,纯化rAAAV感染性滴度为8×10~8TU/ml;用rAAAV转导DF-1细胞,转导后48h用3株IBDV感染,感染后不同时间用TCID_(50)测定法检测感染性病毒,用半定量RT-PCR检测病毒基因表达,。结果显示,在IBDV Lukert株感染后96h,miVP2E和miVP1表达细胞中VP2基因表达抑制率分别为85.2%和89.6%,TCID_(50)分别下降6和7lgs;用另两株IBDV感染miVP2E和miVP1表达细胞,感染性病毒测定结果显示,miVP2E表达细胞中2株IBDV的TCID_(50)分别下降7lgs(YEZ株)和2.5lgs(LYG株),miVP1表达细胞中2株IBDV的TCID_(50)分别下降7lgs(YEZ株)和6.5lgs(LYG株),基因表达和病毒复制抑制作用至少维持6天。
Infectious bursal disease virus(IBDV) is the causative agent of a highly contagious disease in young chickens known as infectious bursal disease(IBD).IBDV infections cause not only different degrees of mortality in chicks,but also vaccination failure to other diseases.Presently,IBD is controlled mainly by vaccination,but its protective effect is compromised by the apoptotic effect of live vaccines on the bursa of the vaccinated chickens and recent emergence of very virulent IBDV strains.Therefore, novel strategies are needed for effective control of the disease.
RNA interference(RNAi) is a post-transcriptional gene silencing mechanism conserved in eukaryotes ranging from worms to humans,which has been shown to be a novel anti-viral strategy for a variety of viral infections.To investigate the feasibility of RNAi technology for suppressing IBDV infection,we in this study used the recently developed RNAi system tailored for chickens to drive miRNA expression.In both reporter vector-transfected and IBDV-infected cells,expressions of the two gene-specific miRNA resulted in significant but incomplete inhibition on VP2 gene expression and/or IBDV replication.These two miRNAs were then delivered by recombinant avian adeno-associated virus,showing significant inhibition on IBDV replication.These data demonstrate the highly effective inhibition of IBDV gene expression and viral replication by miRNAs targeting the VP2 gene.The more detailed experimental findings are summerized as follows.
To investigate whether the human RNA polymerase H1 promoter can efficiently express small interference RNAs(siRNAs) in avian cells,10 siRNAs against green fluorescence protein(GFP) gene were predicted using the web-based siDirect software, one of which was selected for synthesis of short hairpin RNA(shRNA) by PCR.The shRNA was inserted into the RNAi vector pSuper containing the human H1 promoter, resulting in siRNA expression vector pSuper-shRNA.The H1-shRNA cassette was then subcloned into GFP gene expression vector pEGFP-H1,resulting in another expression vector pEGFP-H1-shRNA.The pEGFP-H1-shRNA or pSuper-shRNA+ pEGFP-N1 vector was transfected into simian COS-1 cells,human 293-T cells,chicken embryonic liver(CEL) cells and chicken embryonic fibroblast(CEF) cells and the transfected cells were submitted to fluorescence microscopy.Compared to pEGFP-N1-transfected cells, significant decreases in fluorescence density were evident in the mammalian cells transfected with pEGFP-H1-shRNA or pSuper-shRNA+ peGFP-N1 from 24 h after transfection,which was not seen in the avian cells transfected with the same vector(s). These data demonstrate that human H1 promoter can efficiently transcribe shRNA in the mammalian cells,but the transcription activity in the avian cells is relatively low, suggesting that the promoters of avian origin should be used for expression of shRNA in avian cells.
To verify the miRNA expression system in avian cells using the avian U6 promoter -controlled pRFPRNAiC vector,10 siRNAs against GFP reporter gene were predicted using Genscript software,one ofwhich was selected for double-stranded oligonucleotide synthesis by PCR.The oligonucleotide was inserted into the miRNA expression vector, resulting in the recombinant vector pRFPRNAiGFP,pEGFP-N1 or pRFPRNAiGFP + pEGFP-N1 vector was transfeeted into COS-1 cells,293-T cells,CEL cells and CEF cells,respectively,and the transfected cells were submitted to fluorescence microscopy. The results showed that the total fluorescence of the pRFPRNAiGFP + pEGFP-N1-transfeeted cells was significantly lower than that of pEGFP-N1-transfected cells,especially in avian cells.These data demonstrate that avian U6 promoter can efficiently transcribe shRNAs in both mammalian and avian cells and the pRFPRNAiC vector is a suitable vector for miRNA expression in avian cells.
To investigate the feasibility of inhibiting IBDV replication using the miRNA expression vector pRFPRNAiC,the VP2 gene without its stop codon was amplified from the total RNA extracted from IBDV Lukert strain-infected cells and fused to the N-terminal of GFP gene in pEGFP-N1 vector,resulting in a reporter vector pVP2-EGFP. Five potential miRNAs targeting the VP2 gene of IBDV were selected for double-stranded nucleotide synthesis by PCR and subcloned individually into the pRFPRNAiC vector,resulting in miRNA expression vectors pRFPRNAmiVP2A, pRFPRNAmiVP2B,pRFPRNAmiVP2C,pRFPRNAmiVP2D and pRFPRNAmiVP2E.
Each of the five miRNA expression vectors was co-transfected into DF-1 cells with the reporter vector pVP2-EGFP and expression of the five miRNAs was demonstrated by Northern dot blotting.The transfected cells were first submitted to fluorescence microscopy,showing significant decreases in GFP-positive cell number.Flow cytometry showed significant decrease in total fluorescene ranging from 59.7%to 78.5%.The two more efficient miRNAs,miVP2A and miVP2E,were subcloned into a neo gene-containing RNAi vector individually or in combination and the resulting recombinant vectors were transfected individually into DF-1 cells.After selection with G418 for 1 week,the transfected cells were infected with IBDV Lukert strain,and gene silencing effects on the VP2 expression and viral replication were tested by semi-quantitative RT-PCR and virus titration assay,showing decreases in virus replication by 5-61gs of TCID_(50).These results provide strong evidence that miRNAs targeting the VP2 gene can efficiently inhibit gene expression and/or replication of IBDV.
To further investigate the inhibitory effect of the miRNAs delivered recombinant AAAV,the miVP2E and miVP1(targeting the VP1 gene) together with RFP expression cassette were subcloned individually into AAAV transfer vector pAITR.The resulting vectors were co-transfected individually into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper.The presence of the miRNA expression cassettes in the recombinant AAAV particles was shown by PCR.DF-1 cells in each of 35-mm dishes were transducted with 8×10~8 rAAAV and then infected with 3 different IBDV isolates 48 h after transduction.Ninety six hours after infection, semi-quantitative RT-PCR showed an 85.2%(Lukert stain) decrease of the VP2 transcript in the miVPE-expressing cells,while an 89.6%decrease in the miVP1-expressing cells.Infectious virus titration showed an 61gs(Lukert stain),71gs (YEZ strain) or 2.51gs(LYG Strain) decrease of TCID_(50) in the miVP2E-expressing cells and 71gs(Lukert stain),71gs(YEZ strain) or 6.51gs(LYG Strain) decrease in the miVP1-expressing cells.The inhibitory effect remained for at least 6 days after IBDV infection.
RNA干扰(RNA interference,RNAi)是近年来快速发展的一种转录后基因沉默技术,在功能基因组研究、肿瘤治疗和抗病毒感染等方面具有良好的应用前景,已被证明能抑制几乎所有种属病毒的复制。为探索用RNAi技术抑制IBDV复制的可行性,本研究在比较不同来源RNA聚合酶启动子转录活性基础上,选用禽源U6启动子构建短干扰RNA(short interference RNA,siRNA)表达载体,以绿色荧光蛋白(green fluorescent protein,GFP)基因为报告基因,证明在禽源细胞中能成功实现特异基因表达沉默;进而用专业软件预测IBDV VP2基因特异性siRNA,将人工合成的微小RNA(microRNA,miRNA)插入表达载体,以细胞转染法筛选出2个有效抑制IBDV VP2基因表达的miRNA,将其克隆入含neo基因的表达载体,经G418筛选获得抗性细胞克隆,用感染试验证明能显著抑制IBDV复制;最后将miRNA表达盒插入禽腺联病毒(avian adeno-associated virus,AAAV)转移载体,用重组病毒感染细胞进行的试验结果证明,表达的miRNA对IBDV复制不仅具有更为显著的抑制作用,而且对异源毒株复制也有类似的抑制效果,为IBD防制研究开辟了新的途径,现将试验结果总结如下。
目前siRNA表达试验多用人或鼠源H1或U6启动子在哺乳动物细胞中进行,对于这些启动子能否在禽细胞中有效转录短发夹RNA(small hairpin RNA,shRNA)或miRNA仍有争议。我们利用siDirect软件预测GFP基因特异性siRNA,将人工合成的相应shRNA插入含人H1启动子的pSuper载体,获得重组载体pSuper-shRNA;再将shRNA表达盒插入含GFP基因的pEGFP-N1载体,获得重组载体pGFP-H1-shRNA。分别以pEGFP-N1、pSuper-shRNA+pEGFP-N1和pGFP-H1-shRNA转染哺乳动物源COS-1、293-T细胞和禽源鸡胚肝(CEL)、鸡胚成纤维(CEF)细胞,根据相同条件下转染细胞培养中荧光阳性细胞数及荧光强度的变化,比较人H1启动子在哺乳动物源和禽源细胞中转录shRNA的效率。结果显示,人H1启动子在哺乳动物细胞中能有效转录shRNA,但在禽源细胞中的转录效率很低。这些试验结果表明,开展禽源细胞siRNA表达研究应选用禽源启动子。
为了开展禽源细胞RNAi研究,以含鸡U6启动子的pRFPRNAiC为miRNA表达载体,用Genscript软件预测GFP序列特异性siRNA,从10个潜在的siRNA中随机选择1个siRNA序列,用PCR合成双链寡核苷酸,将其插入表达载体pRFPRNAiC,用获得的重组载体pRFPRNAiGFP与报告质粒pEGFP-N1共转染COS-1、293-T、CEL和CEF细胞,根据相同条件下GFP阳性细胞数及荧光强度变化,比较哺乳动物和禽细胞中鸡U6启动子表达miRNA的效率。结果显示,pRFPRNAiGFP在哺乳动物和禽源细胞中都能表达抑制性miRNA,在禽源细胞中转录效率较高。这些试验结果表明,源禽U6启动子转录miRNA的细胞种属特异性相对较弱,pRFPRNAiC载体可用于禽源细胞的miRNA表达研究。
为了探索用RNAi技术抑制IBDV复制的可行性,利用Genscript软件预测针对IBDV VP2基因的siRNA,从10个潜在的siRNA中选择5个siRNA序列,将PCR产生的双链寡核苷酸插入pRFPRNAiC载体,获得重组载体pRFPRNAmiVP2A、pRFPRNAmiVP2B、pRFPRNAmiVP2C、pRFPRNAmiVP2D、pRFPRNAmiVP2E;根据IBDV Lukert株VP2基因序列设计引物,用PCR扩增不含自身翻译终止码的VP2基因,将扩增产物与pEGFP-N1载体中GFP基因的N端融合,获得报告载体pVP2-GFP。分别将5个miRNA表达载体与报告载体共转染DF-1细胞,在转染后24h用Northern Dot Blotting确认miRNA表达,然后分别用荧光共聚焦显微镜和流式细胞术对转染细胞培养中的荧光阳性细胞数及荧光总量进行定性和定量分析。结果与报告载体单独转染细胞相比,5个miRNA表达载体与报告载体共转染细胞的荧光抑制效率在59.7%-78.5%之间;将抑制效果较好的miVP2A和miVP2E表达盒分别克隆入含neo基因的pTarget载体,用获得的重组载体转染DF-1细胞,经G418筛选后获得抗性细胞,用IBDV Lurket感染,感染三天后用半定量RT-PCR测定VP2基因表达,并测定IBDV半数组织细胞感染剂量(TCID_(50))。结果显示,miVP2A和miVP2E表达细胞中VP2基因表达抑制率分别为80.7%和75.0%,TCID_(50)分别下降6和5lgs。这些试验结果表明,针对VP2基因的两个miRNA对IBDV基因表达和复制均有显著的抑制作用。
利用重组AAAV稳定表达外源基因的特点,用限制酶消化法去除含AAAV全基因组质粒载体pCR-AAAV中的Pep和Cap蛋白编码序列,获得AAAV转移载体pAITR;分别将miVP2E和针对VP1基因的miVP1表达盒连同红色荧光蛋白(redfluorescent protein,RFP)基因表达盒插入pAITR中左、右ITR之间,分别用获得的重组载体pAITR-RFP-miVP2E和pAITR-RFP-miVP1与AAAV包装载体pcDNA-ARC及腺病毒辅助载体pHelper共转染AAV-293细胞,经PCR检测证明获得的rAAAV中含有miRNA表达盒,纯化rAAAV感染性滴度为8×10~8TU/ml;用rAAAV转导DF-1细胞,转导后48h用3株IBDV感染,感染后不同时间用TCID_(50)测定法检测感染性病毒,用半定量RT-PCR检测病毒基因表达,。结果显示,在IBDV Lukert株感染后96h,miVP2E和miVP1表达细胞中VP2基因表达抑制率分别为85.2%和89.6%,TCID_(50)分别下降6和7lgs;用另两株IBDV感染miVP2E和miVP1表达细胞,感染性病毒测定结果显示,miVP2E表达细胞中2株IBDV的TCID_(50)分别下降7lgs(YEZ株)和2.5lgs(LYG株),miVP1表达细胞中2株IBDV的TCID_(50)分别下降7lgs(YEZ株)和6.5lgs(LYG株),基因表达和病毒复制抑制作用至少维持6天。
Infectious bursal disease virus(IBDV) is the causative agent of a highly contagious disease in young chickens known as infectious bursal disease(IBD).IBDV infections cause not only different degrees of mortality in chicks,but also vaccination failure to other diseases.Presently,IBD is controlled mainly by vaccination,but its protective effect is compromised by the apoptotic effect of live vaccines on the bursa of the vaccinated chickens and recent emergence of very virulent IBDV strains.Therefore, novel strategies are needed for effective control of the disease.
RNA interference(RNAi) is a post-transcriptional gene silencing mechanism conserved in eukaryotes ranging from worms to humans,which has been shown to be a novel anti-viral strategy for a variety of viral infections.To investigate the feasibility of RNAi technology for suppressing IBDV infection,we in this study used the recently developed RNAi system tailored for chickens to drive miRNA expression.In both reporter vector-transfected and IBDV-infected cells,expressions of the two gene-specific miRNA resulted in significant but incomplete inhibition on VP2 gene expression and/or IBDV replication.These two miRNAs were then delivered by recombinant avian adeno-associated virus,showing significant inhibition on IBDV replication.These data demonstrate the highly effective inhibition of IBDV gene expression and viral replication by miRNAs targeting the VP2 gene.The more detailed experimental findings are summerized as follows.
To investigate whether the human RNA polymerase H1 promoter can efficiently express small interference RNAs(siRNAs) in avian cells,10 siRNAs against green fluorescence protein(GFP) gene were predicted using the web-based siDirect software, one of which was selected for synthesis of short hairpin RNA(shRNA) by PCR.The shRNA was inserted into the RNAi vector pSuper containing the human H1 promoter, resulting in siRNA expression vector pSuper-shRNA.The H1-shRNA cassette was then subcloned into GFP gene expression vector pEGFP-H1,resulting in another expression vector pEGFP-H1-shRNA.The pEGFP-H1-shRNA or pSuper-shRNA+ pEGFP-N1 vector was transfected into simian COS-1 cells,human 293-T cells,chicken embryonic liver(CEL) cells and chicken embryonic fibroblast(CEF) cells and the transfected cells were submitted to fluorescence microscopy.Compared to pEGFP-N1-transfected cells, significant decreases in fluorescence density were evident in the mammalian cells transfected with pEGFP-H1-shRNA or pSuper-shRNA+ peGFP-N1 from 24 h after transfection,which was not seen in the avian cells transfected with the same vector(s). These data demonstrate that human H1 promoter can efficiently transcribe shRNA in the mammalian cells,but the transcription activity in the avian cells is relatively low, suggesting that the promoters of avian origin should be used for expression of shRNA in avian cells.
To verify the miRNA expression system in avian cells using the avian U6 promoter -controlled pRFPRNAiC vector,10 siRNAs against GFP reporter gene were predicted using Genscript software,one ofwhich was selected for double-stranded oligonucleotide synthesis by PCR.The oligonucleotide was inserted into the miRNA expression vector, resulting in the recombinant vector pRFPRNAiGFP,pEGFP-N1 or pRFPRNAiGFP + pEGFP-N1 vector was transfeeted into COS-1 cells,293-T cells,CEL cells and CEF cells,respectively,and the transfected cells were submitted to fluorescence microscopy. The results showed that the total fluorescence of the pRFPRNAiGFP + pEGFP-N1-transfeeted cells was significantly lower than that of pEGFP-N1-transfected cells,especially in avian cells.These data demonstrate that avian U6 promoter can efficiently transcribe shRNAs in both mammalian and avian cells and the pRFPRNAiC vector is a suitable vector for miRNA expression in avian cells.
To investigate the feasibility of inhibiting IBDV replication using the miRNA expression vector pRFPRNAiC,the VP2 gene without its stop codon was amplified from the total RNA extracted from IBDV Lukert strain-infected cells and fused to the N-terminal of GFP gene in pEGFP-N1 vector,resulting in a reporter vector pVP2-EGFP. Five potential miRNAs targeting the VP2 gene of IBDV were selected for double-stranded nucleotide synthesis by PCR and subcloned individually into the pRFPRNAiC vector,resulting in miRNA expression vectors pRFPRNAmiVP2A, pRFPRNAmiVP2B,pRFPRNAmiVP2C,pRFPRNAmiVP2D and pRFPRNAmiVP2E.
Each of the five miRNA expression vectors was co-transfected into DF-1 cells with the reporter vector pVP2-EGFP and expression of the five miRNAs was demonstrated by Northern dot blotting.The transfected cells were first submitted to fluorescence microscopy,showing significant decreases in GFP-positive cell number.Flow cytometry showed significant decrease in total fluorescene ranging from 59.7%to 78.5%.The two more efficient miRNAs,miVP2A and miVP2E,were subcloned into a neo gene-containing RNAi vector individually or in combination and the resulting recombinant vectors were transfected individually into DF-1 cells.After selection with G418 for 1 week,the transfected cells were infected with IBDV Lukert strain,and gene silencing effects on the VP2 expression and viral replication were tested by semi-quantitative RT-PCR and virus titration assay,showing decreases in virus replication by 5-61gs of TCID_(50).These results provide strong evidence that miRNAs targeting the VP2 gene can efficiently inhibit gene expression and/or replication of IBDV.
To further investigate the inhibitory effect of the miRNAs delivered recombinant AAAV,the miVP2E and miVP1(targeting the VP1 gene) together with RFP expression cassette were subcloned individually into AAAV transfer vector pAITR.The resulting vectors were co-transfected individually into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper.The presence of the miRNA expression cassettes in the recombinant AAAV particles was shown by PCR.DF-1 cells in each of 35-mm dishes were transducted with 8×10~8 rAAAV and then infected with 3 different IBDV isolates 48 h after transduction.Ninety six hours after infection, semi-quantitative RT-PCR showed an 85.2%(Lukert stain) decrease of the VP2 transcript in the miVPE-expressing cells,while an 89.6%decrease in the miVP1-expressing cells.Infectious virus titration showed an 61gs(Lukert stain),71gs (YEZ strain) or 2.51gs(LYG Strain) decrease of TCID_(50) in the miVP2E-expressing cells and 71gs(Lukert stain),71gs(YEZ strain) or 6.51gs(LYG Strain) decrease in the miVP1-expressing cells.The inhibitory effect remained for at least 6 days after IBDV infection.
引文
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