单链抗体介导狂犬病毒shRNA靶向制剂的研究
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摘要
狂犬病是由狂犬病病毒引起的一种动物源性人兽共患传染病,病死率极高,在世界范围内广泛流行,我国是狂犬病流行最严重的国家之一。人被带狂犬病毒的犬咬伤后,控制的基本策略是暴露后治疗(Post exposure treatment, PET),高危暴露(Ⅲ级)时,WHO推荐使用抗狂犬病免疫球蛋白作为被动免疫制剂。动物源免疫球蛋白可能导致轻重不等的过敏反应,而人抗狂犬病免疫球蛋白又有传播血液疾病的危险。因此,寻找有效和特异的治疗狂犬病的新方法显得十分重要。
RNA干扰(RNA interference, RNAi)是内源性或外源性双链RNA (double stranded RNA, dsRNA)诱导的mRNA水平的基因表达沉默现象,将21-23nt的干扰性双链小RNA (short interfering RNA, siRNA)导入目的细胞,可以诱导高效且特异的RNAi效应。目前,RNAi已经成为替代基因敲除技术进行基因功能研究的有力工具。随着RNAi机制及其应用研究的不断深入,在癌症和一些病毒性疾病如艾滋病、乙型肝炎等,用siRNA治疗研究已进入一期或二期临床试验,有望成为新型的治疗制剂。
RNA干扰可由人工合成双股siRNA或者将表达shRNA (small hairpin RNA, shRNA)的载体转入细胞中,在Dicer酶作用下,shRNA被加工成siRNA。由于体外合成的siRNA导入机体后易被降解,而shRNA良好的重复性和较长的细胞内效应期,价格经济,也是以siRNA进行基因治疗的首选方式。所以本研究选择以质粒载体表达shRNA进行靶向药物效果评价。
治疗性小干扰RNA面临最大的问题是在体内安全运送而不被RNA酶、内质网的滞留作用、肾脏代谢等复杂的体内降解消耗机制作用而减少或消失,目前解决这个难题的有效方法之一是将siRNA用能识别感染细胞表面特异受体的蛋白与siRNA作用,将siRNA包裹起来,既保护了siRNA又使其实现靶向运送。体内细胞类型特异性的基因沉默可以通过siRNA与细胞类型特异性亲和配体或单克隆抗体结合而实现。即抗体指导的siRNA复合物通过内吞作用进入靶细胞,随后释放到细胞浆,通过RNA干扰路径,特异性地沉默靶基因的表达。抗体成份与小核酸分子结合后能在体内实现有效运送siRNA。已在体内证明了这种运送方式不使机体产生特异免疫反应和毒性,成为有前景的治疗性方法。
本研究以针对人源狂犬病毒G糖蛋白二硫键稳定单链抗体(single chain disulfide-stabilized antibody, scdsFv)来特异识别感染细胞表面出芽的狂犬病毒,用绿脓杆菌外毒素跨膜区ETA部分实现穿膜,以酵母DNA结合结构域GAL4与含有特异shRNA的质粒结合,制备了狂犬病毒靶向药物,并对该药物在体内外抑制RV的复制情况进行了探索,以期为狂犬病暴露后乃至发病后的治疗积累必要的实验数据。
本研究针对狂犬病毒N蛋白编码基因,设计合成了4对shRNA,以随机序列shRNA为阴性对照,将shRNA分别连接到表达载体pRNATU6.3上,转染BHK-21细胞后,在潮霉素抗性压力下,筛选获得稳定表达shRNA的BHK-21细胞株。在体外检测其对狂犬病毒标准攻击毒CVS-11的抑制效果,用直接免疫荧光、荧光定量PCR、Western blot检测表明,筛选出具有高效抑制RV复制的2个靶位点N-489和N-701。
为获得能良好识别RV感染细胞的单链抗体,根据已发表的具有广谱中和效力的人源RVG单抗序列,设计点突变位点,人工合成scdsFv序列,连接到pET-22b (+)载体上,经大肠杆菌表达,获得包涵体表达的scdsFv蛋白。亲和力、特异性和中和能力检测表明该scdsFv具有特异识别狂犬病毒感染细胞的能力。
为了制备能与含shRNA的质粒载体结合的具有导向作用的嵌合蛋白,设计引物从质粒PE40-GAL4-T上扩增获得了绿脓杆菌外毒素穿细胞膜区-酵母DNA结合结构域ETA-GAL4基因,将ETA-GAL4、scdsFv通过搭桥PCR,获得了scdsFv-ETA-GAL4基因,即SEG,在原核表达载体pET28a中表达了融合蛋白SEG。此蛋白以包涵体表达为主,经包涵体变性、Ni-柱纯化、复性后获得有活性SEG蛋白,实验表明此融合蛋白明显保留有抗原结合活性,可特异结合细胞膜上的病毒抗原。
SEG可以结合并转运shRNA到狂犬病毒感染细胞。凝胶阻滞实验检测表明,SEG蛋白具有与shRNA结合的能力,且这种结合存在着剂量依赖关系：1nM SEG可以结合约10nM shRNA;绿色荧光蛋白(GFP)显示SEG蛋白可将含shRNA的质粒特异性运送到病毒感染的细胞。MTT检测表明SEG-shRNA复合物对细胞生长没有毒性作用。在感染细胞中,病毒RNA水平和蛋白水平检测结果均表明,SEG靶向转运shRNA能够有效地抑制病毒的复制。
在小鼠后肢肌肉注射CVS-24毒以制备动物模型。在小鼠感染模型中,取50LD50 RV标准攻击毒CVS-24攻毒后,尾静脉注射SEG-shRNA复合物,进行狂犬病毒的体内靶向效应试验。结果显示,靶向药物SEG-shRNA注射后30h,流式细胞仪检测表明仅在注射病毒的右侧后腿肌肉检测到GFP,其余组织和内脏器官均无GFP表达。表明此复合物在体内可实现靶向性运送。小鼠保护性试验表明,注射病毒后8h尾静脉注射SEG-shRNA,5d后检测小鼠脑内RV含量：RT-PCR、Western blot检测表明使用靶向药物组RV含量明显少于病毒对照组,条带亮度变弱；qRT-PCR表明靶向药物组比病毒对照减少4.88倍；小鼠发病时间比病毒对照组晚48h,用SEG-shRNA组动物存活率达50%,而病毒对照100%死亡。检测小鼠体内IFN-a未见升高,与正常组差异不明显,证明在动物体内产生的保护作用是由针对病毒RNA的特异shRNA所介导。结果表明SEG-shRNA对小鼠有明显保护作用。总之,试验结果显示,SEG蛋白可以特异性转运含shRNA质粒进入细胞,并沉默靶基因的表达,因此可以用于狂犬病毒感染的特异辅助性救治研究。
Rabies is a lethal zoonotic infection disease induced by Rabies virus and its case fatality ratio (CFR) is 100 percent approximately. China is one of the most severe regions of rabies spread. According to the recommendations of WHO rabies experts committee, post-exposure treatment (PET) must be taken as soon as possible after a bite or a scratch occurred. Immunoglobulin is one of the regents in PET, but the zoogenous immunoglobulin is a kind of allergen, and the products made from human blood are infective agents of some body fluid transmitted diseases. Thus, new antiviral approaches that allow efficient and specific destruction of rabies virus are required.
RNA interference (RNAi) refers to the small dsRNA-guided gene silencing phenomenon conserved in a wide range of eukaryotic organisms from plants to mammals. It is an important mechanism of cellular defense and differentiation regulation and plays an important role in the regulation of gene expression, the prevention of viral infection and the control of gene transposition. RNAi in target cells could be induced specifically and effectively by 21nt to 23nt short interfering RNA (siRNA) and has become a powerful tool to explore gene functions replacing the knock-out technique. With the progressions of the research about RNAi, some investigations have been performed successfully to inhibit the replication of viruses.
RNAi can be induced by the introduction of synthesized double-stranded siRNA or by the intracellular generation of siRNA from the vector-driven expression of precursor small hairpin RNA (shRNA) which is subsequently processed in the cytoplasm by Dicer into siRNA. Synthesized siRNA has a very short half-life of a few minutes in vivo. The gene-silencing effects of the shRNA are long-lasting and reproducible which make the results more reliability, and it can be prepared and produced large-scale at low cost. ShRNA seems to be ideal for delivering of siRNA in vivo. In this study we selected expression plasmids for shRNA to research targeted inhibition rabies virus replication.
The field of RNAi is moving forward at a remarkable pace.Because of their ability to induce transient and reversible effects, siRNAs offer a drug-like approach to disease treatment and thus, several clinical trails are being inducted to assess the safety and efficacy of this approach.
A major challenge to the development of RNAi-based therapeutics is specific and efficient in vivo delivery to target cells. Naked siRNA has a very short half-life of a few minutes in serum owing to degradation by ribonucleases (RNAase), rapid renal excretion, uptake by the reticuloendothelial system (RES) and aggregation with serum proteins. Recent studies suggest that cell type-specific gene silencing in vivo can be achieved by combining therapeutic RNAi with cell type-specific affinity ligands or monoclonal antibodies. The antibody-directed siRNA complex enters target cells through endocytosis and is subsequently released to the cytosol to specifically silence target gene expression through biologically conserved RNAi pathways. Antibody fragments fused with a small basic nucleic acid-binding protein is an effective delivery vehicle in vivo. The demonstrated specificity of in vivo gene silencing and the lack of nonspecific immune activation and systemic toxicity encourage further delivery of therapeutic RNAi.
Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis. The protein consists of three functional domains:1)a RV G-specific single chain disulfide-stabilized antibody confers target cell specificity.2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enables sequence-specific high bacterial lysates displayed both RV infected cells specific and DNA sequence-specific binding in vitro and in vivo. We successful obtain the SEG-shRNA complexes, which targeted the RV infected cells. In this thesis, the replication of rabies virus was inhibited by SEG-shRNA complexes in vivo and in vitro experimentally in order to accumulate essential data of single-chain Fv antibody (scFv) mediated shRNA targeted therapy in the research of rabies virus the PET.
Targeting N gene of RV, four shRNA were designed and constructed based on the vector pRNATU6.3-Hygro which expresses fusion protein of green fluorescent protein (GFP) as a report gene. Four cell strains (N1, N2, N3, N4) expressing the short hairpin RNAs (shRNA) were obtained after the plasmids were transfected into the BHK-21 cell line and screened under the pressure of Hygromycin B (300μg/ml).These cell strains were infected rabies virus CVS-11 strain respectively, the virus replication were detected and evaluated by directed immunofluorescence assay (DFA) and real-time PCR and 50% tissue culture infective dose (TC1D50). The three detected results were coincidence. We selected effective inhibition RV siRNA targeting N gene, in which N-489 and N-701 were proved that they can specifically inhibit the virus production in BHK-21 cells.
To produce a scFv which can specific recognize the RV infected cells, the heavy (VH) and light chain variable region (VL) sequence of monoclonal antibody SO57 which obtained from Genbank were assembled into a scFv gene using a linker sequence. To construct the recombinant human anti-rabies virus single-chain disulfide-stabilized Fv (scdsFv), Cys sites were introduced into framework region (FR) of VH and VL gene using genetic point mutation technology. The scdsFv gene was cloned into expression vector pET-22b (+) and transformed into E.coli BL21(DE3). The target protein was expressed in form of inclusion bodies. The avidity, specificity characteristics and neutralization capacity of scdsFv showed that scdsFv could bind antigen specifically.
In order to prepare the chimeric protein which could realize targeting transfer DNA which express shRNA into the RV infected cells, rabies virus scdsFv(G) gene and ETA-GAL4 gene were amplified by PCR from vector scdsFv(G)-T and PE40-GAL4-T respectively. Then, the chimeric gene scdsFv(G)-ETA-GAL4 (SEG) was obtained by lapextension PCR and cloned into the prokaryotic expression vector pET28a(+). Recombinant expression plasmid of pET28a(+)-scdsFv(G)-ETA-GAL4 (pET28a-SEG) was transformed into the competent E.coli BL21(DE3) cells for expression under the induction of IPTG. The SEG proteins mainly expressed to form inclusion bodies. The SEG proteins were purified by Ni-NTA column and renatured to obtain the biological activity. The experiment results showed that the fusion protein SEG could bind affinity to RV and the RV infected cells.
We next tested whether SEG could bind and deliver shRNA to RV-infected cells and found that the fusion protein was able to bind shRNA in a dose-dependent manner in a gel-shift assay and 1nM SEG can bind 10nM shRNA molecules. The fusion protein SEG with plasmid pRNATU6.3-shRNA were added to BHK-21 cells culture medium that infected with RV and then green fluorescent protein (GFP) was observed after 24 hours. It was showed that SEG could realize targeting transfer DNA which expressed shRNA into the RV infected cells. Cell viability caused by SEG-shRNA complexes was also determined by MTT assay and the result confirmed that the complexes were almost non-toxic to cell viability. The levels of viral particles from SEG-shRNA-treated infected cells was reduced as demonstrated by RV mRNA and proteins. These results demonstrated that the complex effectively inhibited RV replication in BHK-21 cells.
Because there is no good mouse model for rabies virus, we injected mice intramuscular with CVS-24 as a RV infected animal model to test the ability of SEG fusion protein to specifically deliver shRNA into RV-infected cell in vivo.50LD50 RV-infected mice were injected intravenous with complexes SEG-shRNA. Single-cell suspensions from the hind lambs were examined by flow cytometry, which showed that SEG could specific delivery of shRNA into RV-infected hind lamb cells. The results showed SEG protein could deliver shRNA to RV infected cells specifically. To evaluate the therapeutic potential of antibody-mediated shRNA delivery, we injected SEG-shRNA intravenously 8h after the mice infected lethal dose RV CVS-24. On five days when the RV in brain tissue of mice were detected by RT-PCR and western blot shown that mice treated with SEG-shRNA had lower RV RNA and protein levels than untreated controls. Real-time PCR showed that RV was reduced about 4.88 fold compared to the mock cells. The incubation period of the challenged mice were prolonged significantly (P＜0.01). Evaluation of mouse survival of RV-infected mice showed a significant protection from RV infection by SEG-shRNA treatment. The survival was up to 50% while the virus control all died.
To rule out the possibility that non-specific interferon (IFN) production mediated the protection, we measured serum IFN levels after administration of SEG-shRNA, and found that IFN was not induced in SEG-shRNA treated animals. Taken together, our results showed that SGE enables the specific delivery of shRNA to silence gene expression and SEG-shRNA can be used for adjutant treatment for rabies.
RNA干扰(RNA interference, RNAi)是内源性或外源性双链RNA (double stranded RNA, dsRNA)诱导的mRNA水平的基因表达沉默现象,将21-23nt的干扰性双链小RNA (short interfering RNA, siRNA)导入目的细胞,可以诱导高效且特异的RNAi效应。目前,RNAi已经成为替代基因敲除技术进行基因功能研究的有力工具。随着RNAi机制及其应用研究的不断深入,在癌症和一些病毒性疾病如艾滋病、乙型肝炎等,用siRNA治疗研究已进入一期或二期临床试验,有望成为新型的治疗制剂。
RNA干扰可由人工合成双股siRNA或者将表达shRNA (small hairpin RNA, shRNA)的载体转入细胞中,在Dicer酶作用下,shRNA被加工成siRNA。由于体外合成的siRNA导入机体后易被降解,而shRNA良好的重复性和较长的细胞内效应期,价格经济,也是以siRNA进行基因治疗的首选方式。所以本研究选择以质粒载体表达shRNA进行靶向药物效果评价。
治疗性小干扰RNA面临最大的问题是在体内安全运送而不被RNA酶、内质网的滞留作用、肾脏代谢等复杂的体内降解消耗机制作用而减少或消失,目前解决这个难题的有效方法之一是将siRNA用能识别感染细胞表面特异受体的蛋白与siRNA作用,将siRNA包裹起来,既保护了siRNA又使其实现靶向运送。体内细胞类型特异性的基因沉默可以通过siRNA与细胞类型特异性亲和配体或单克隆抗体结合而实现。即抗体指导的siRNA复合物通过内吞作用进入靶细胞,随后释放到细胞浆,通过RNA干扰路径,特异性地沉默靶基因的表达。抗体成份与小核酸分子结合后能在体内实现有效运送siRNA。已在体内证明了这种运送方式不使机体产生特异免疫反应和毒性,成为有前景的治疗性方法。
本研究以针对人源狂犬病毒G糖蛋白二硫键稳定单链抗体(single chain disulfide-stabilized antibody, scdsFv)来特异识别感染细胞表面出芽的狂犬病毒,用绿脓杆菌外毒素跨膜区ETA部分实现穿膜,以酵母DNA结合结构域GAL4与含有特异shRNA的质粒结合,制备了狂犬病毒靶向药物,并对该药物在体内外抑制RV的复制情况进行了探索,以期为狂犬病暴露后乃至发病后的治疗积累必要的实验数据。
本研究针对狂犬病毒N蛋白编码基因,设计合成了4对shRNA,以随机序列shRNA为阴性对照,将shRNA分别连接到表达载体pRNATU6.3上,转染BHK-21细胞后,在潮霉素抗性压力下,筛选获得稳定表达shRNA的BHK-21细胞株。在体外检测其对狂犬病毒标准攻击毒CVS-11的抑制效果,用直接免疫荧光、荧光定量PCR、Western blot检测表明,筛选出具有高效抑制RV复制的2个靶位点N-489和N-701。
为获得能良好识别RV感染细胞的单链抗体,根据已发表的具有广谱中和效力的人源RVG单抗序列,设计点突变位点,人工合成scdsFv序列,连接到pET-22b (+)载体上,经大肠杆菌表达,获得包涵体表达的scdsFv蛋白。亲和力、特异性和中和能力检测表明该scdsFv具有特异识别狂犬病毒感染细胞的能力。
为了制备能与含shRNA的质粒载体结合的具有导向作用的嵌合蛋白,设计引物从质粒PE40-GAL4-T上扩增获得了绿脓杆菌外毒素穿细胞膜区-酵母DNA结合结构域ETA-GAL4基因,将ETA-GAL4、scdsFv通过搭桥PCR,获得了scdsFv-ETA-GAL4基因,即SEG,在原核表达载体pET28a中表达了融合蛋白SEG。此蛋白以包涵体表达为主,经包涵体变性、Ni-柱纯化、复性后获得有活性SEG蛋白,实验表明此融合蛋白明显保留有抗原结合活性,可特异结合细胞膜上的病毒抗原。
SEG可以结合并转运shRNA到狂犬病毒感染细胞。凝胶阻滞实验检测表明,SEG蛋白具有与shRNA结合的能力,且这种结合存在着剂量依赖关系：1nM SEG可以结合约10nM shRNA;绿色荧光蛋白(GFP)显示SEG蛋白可将含shRNA的质粒特异性运送到病毒感染的细胞。MTT检测表明SEG-shRNA复合物对细胞生长没有毒性作用。在感染细胞中,病毒RNA水平和蛋白水平检测结果均表明,SEG靶向转运shRNA能够有效地抑制病毒的复制。
在小鼠后肢肌肉注射CVS-24毒以制备动物模型。在小鼠感染模型中,取50LD50 RV标准攻击毒CVS-24攻毒后,尾静脉注射SEG-shRNA复合物,进行狂犬病毒的体内靶向效应试验。结果显示,靶向药物SEG-shRNA注射后30h,流式细胞仪检测表明仅在注射病毒的右侧后腿肌肉检测到GFP,其余组织和内脏器官均无GFP表达。表明此复合物在体内可实现靶向性运送。小鼠保护性试验表明,注射病毒后8h尾静脉注射SEG-shRNA,5d后检测小鼠脑内RV含量：RT-PCR、Western blot检测表明使用靶向药物组RV含量明显少于病毒对照组,条带亮度变弱；qRT-PCR表明靶向药物组比病毒对照减少4.88倍；小鼠发病时间比病毒对照组晚48h,用SEG-shRNA组动物存活率达50%,而病毒对照100%死亡。检测小鼠体内IFN-a未见升高,与正常组差异不明显,证明在动物体内产生的保护作用是由针对病毒RNA的特异shRNA所介导。结果表明SEG-shRNA对小鼠有明显保护作用。总之,试验结果显示,SEG蛋白可以特异性转运含shRNA质粒进入细胞,并沉默靶基因的表达,因此可以用于狂犬病毒感染的特异辅助性救治研究。
Rabies is a lethal zoonotic infection disease induced by Rabies virus and its case fatality ratio (CFR) is 100 percent approximately. China is one of the most severe regions of rabies spread. According to the recommendations of WHO rabies experts committee, post-exposure treatment (PET) must be taken as soon as possible after a bite or a scratch occurred. Immunoglobulin is one of the regents in PET, but the zoogenous immunoglobulin is a kind of allergen, and the products made from human blood are infective agents of some body fluid transmitted diseases. Thus, new antiviral approaches that allow efficient and specific destruction of rabies virus are required.
RNA interference (RNAi) refers to the small dsRNA-guided gene silencing phenomenon conserved in a wide range of eukaryotic organisms from plants to mammals. It is an important mechanism of cellular defense and differentiation regulation and plays an important role in the regulation of gene expression, the prevention of viral infection and the control of gene transposition. RNAi in target cells could be induced specifically and effectively by 21nt to 23nt short interfering RNA (siRNA) and has become a powerful tool to explore gene functions replacing the knock-out technique. With the progressions of the research about RNAi, some investigations have been performed successfully to inhibit the replication of viruses.
RNAi can be induced by the introduction of synthesized double-stranded siRNA or by the intracellular generation of siRNA from the vector-driven expression of precursor small hairpin RNA (shRNA) which is subsequently processed in the cytoplasm by Dicer into siRNA. Synthesized siRNA has a very short half-life of a few minutes in vivo. The gene-silencing effects of the shRNA are long-lasting and reproducible which make the results more reliability, and it can be prepared and produced large-scale at low cost. ShRNA seems to be ideal for delivering of siRNA in vivo. In this study we selected expression plasmids for shRNA to research targeted inhibition rabies virus replication.
The field of RNAi is moving forward at a remarkable pace.Because of their ability to induce transient and reversible effects, siRNAs offer a drug-like approach to disease treatment and thus, several clinical trails are being inducted to assess the safety and efficacy of this approach.
A major challenge to the development of RNAi-based therapeutics is specific and efficient in vivo delivery to target cells. Naked siRNA has a very short half-life of a few minutes in serum owing to degradation by ribonucleases (RNAase), rapid renal excretion, uptake by the reticuloendothelial system (RES) and aggregation with serum proteins. Recent studies suggest that cell type-specific gene silencing in vivo can be achieved by combining therapeutic RNAi with cell type-specific affinity ligands or monoclonal antibodies. The antibody-directed siRNA complex enters target cells through endocytosis and is subsequently released to the cytosol to specifically silence target gene expression through biologically conserved RNAi pathways. Antibody fragments fused with a small basic nucleic acid-binding protein is an effective delivery vehicle in vivo. The demonstrated specificity of in vivo gene silencing and the lack of nonspecific immune activation and systemic toxicity encourage further delivery of therapeutic RNAi.
Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis. The protein consists of three functional domains:1)a RV G-specific single chain disulfide-stabilized antibody confers target cell specificity.2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enables sequence-specific high bacterial lysates displayed both RV infected cells specific and DNA sequence-specific binding in vitro and in vivo. We successful obtain the SEG-shRNA complexes, which targeted the RV infected cells. In this thesis, the replication of rabies virus was inhibited by SEG-shRNA complexes in vivo and in vitro experimentally in order to accumulate essential data of single-chain Fv antibody (scFv) mediated shRNA targeted therapy in the research of rabies virus the PET.
Targeting N gene of RV, four shRNA were designed and constructed based on the vector pRNATU6.3-Hygro which expresses fusion protein of green fluorescent protein (GFP) as a report gene. Four cell strains (N1, N2, N3, N4) expressing the short hairpin RNAs (shRNA) were obtained after the plasmids were transfected into the BHK-21 cell line and screened under the pressure of Hygromycin B (300μg/ml).These cell strains were infected rabies virus CVS-11 strain respectively, the virus replication were detected and evaluated by directed immunofluorescence assay (DFA) and real-time PCR and 50% tissue culture infective dose (TC1D50). The three detected results were coincidence. We selected effective inhibition RV siRNA targeting N gene, in which N-489 and N-701 were proved that they can specifically inhibit the virus production in BHK-21 cells.
To produce a scFv which can specific recognize the RV infected cells, the heavy (VH) and light chain variable region (VL) sequence of monoclonal antibody SO57 which obtained from Genbank were assembled into a scFv gene using a linker sequence. To construct the recombinant human anti-rabies virus single-chain disulfide-stabilized Fv (scdsFv), Cys sites were introduced into framework region (FR) of VH and VL gene using genetic point mutation technology. The scdsFv gene was cloned into expression vector pET-22b (+) and transformed into E.coli BL21(DE3). The target protein was expressed in form of inclusion bodies. The avidity, specificity characteristics and neutralization capacity of scdsFv showed that scdsFv could bind antigen specifically.
In order to prepare the chimeric protein which could realize targeting transfer DNA which express shRNA into the RV infected cells, rabies virus scdsFv(G) gene and ETA-GAL4 gene were amplified by PCR from vector scdsFv(G)-T and PE40-GAL4-T respectively. Then, the chimeric gene scdsFv(G)-ETA-GAL4 (SEG) was obtained by lapextension PCR and cloned into the prokaryotic expression vector pET28a(+). Recombinant expression plasmid of pET28a(+)-scdsFv(G)-ETA-GAL4 (pET28a-SEG) was transformed into the competent E.coli BL21(DE3) cells for expression under the induction of IPTG. The SEG proteins mainly expressed to form inclusion bodies. The SEG proteins were purified by Ni-NTA column and renatured to obtain the biological activity. The experiment results showed that the fusion protein SEG could bind affinity to RV and the RV infected cells.
We next tested whether SEG could bind and deliver shRNA to RV-infected cells and found that the fusion protein was able to bind shRNA in a dose-dependent manner in a gel-shift assay and 1nM SEG can bind 10nM shRNA molecules. The fusion protein SEG with plasmid pRNATU6.3-shRNA were added to BHK-21 cells culture medium that infected with RV and then green fluorescent protein (GFP) was observed after 24 hours. It was showed that SEG could realize targeting transfer DNA which expressed shRNA into the RV infected cells. Cell viability caused by SEG-shRNA complexes was also determined by MTT assay and the result confirmed that the complexes were almost non-toxic to cell viability. The levels of viral particles from SEG-shRNA-treated infected cells was reduced as demonstrated by RV mRNA and proteins. These results demonstrated that the complex effectively inhibited RV replication in BHK-21 cells.
Because there is no good mouse model for rabies virus, we injected mice intramuscular with CVS-24 as a RV infected animal model to test the ability of SEG fusion protein to specifically deliver shRNA into RV-infected cell in vivo.50LD50 RV-infected mice were injected intravenous with complexes SEG-shRNA. Single-cell suspensions from the hind lambs were examined by flow cytometry, which showed that SEG could specific delivery of shRNA into RV-infected hind lamb cells. The results showed SEG protein could deliver shRNA to RV infected cells specifically. To evaluate the therapeutic potential of antibody-mediated shRNA delivery, we injected SEG-shRNA intravenously 8h after the mice infected lethal dose RV CVS-24. On five days when the RV in brain tissue of mice were detected by RT-PCR and western blot shown that mice treated with SEG-shRNA had lower RV RNA and protein levels than untreated controls. Real-time PCR showed that RV was reduced about 4.88 fold compared to the mock cells. The incubation period of the challenged mice were prolonged significantly (P＜0.01). Evaluation of mouse survival of RV-infected mice showed a significant protection from RV infection by SEG-shRNA treatment. The survival was up to 50% while the virus control all died.
To rule out the possibility that non-specific interferon (IFN) production mediated the protection, we measured serum IFN levels after administration of SEG-shRNA, and found that IFN was not induced in SEG-shRNA treated animals. Taken together, our results showed that SGE enables the specific delivery of shRNA to silence gene expression and SEG-shRNA can be used for adjutant treatment for rabies.
引文
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