携带人S100A1基因的重组腺相关病毒载体的构建及鉴定

目的:构建携带有人S100A1基因的重组腺相关病毒(Adeno-associated viru,AAV)载体,并对其进行鉴定.方法:用BamH I和EcoRI 将目的基因pUC57-Simple-S100A1和腺相关病毒骨架质粒pAAV-IRES-ZsGreen1行双酶切,回收酶切质粒的目的片段进行连接,产物转化感受态DH5a,获得重组腺相关病毒骨架质粒pAAV-IRES-ZsGreen1-S100A1.酶切及测序鉴定后,将pAAV-IRES-ZsGreen1-S100A1、包装质粒pAAV-RC和辅助质粒pHelper三质粒共转染AAV-293,包装重组腺相关病毒rAAV-IRES-ZsGreen1-S100A1.收获重组病毒后感染HEK293细胞,荧光计数法测定病毒滴度,病毒基因组外源基因扩增鉴定重组病毒的包装是否成功.结果:重组腺相关病毒骨架质粒pAAV-IRES-ZsGreen1-S100A1经双酶切和测序鉴定正确;荧光显微镜下观察转染AAV-293细胞72 h后,病毒包装效率达95％～100％,荧光计数法测定病毒感染滴度达(2～3)×107 TU/mL;提取重组病毒基因组成功扩增出外源目的基因S100A1片段.结论:成功构建携带有人S100A1的重组腺相关病毒载体rAAV-IRES-ZsGreen1-S100A1,收获的病毒具有较高滴度,为今后利用腺相关病毒载体进行S100A1基因转染治疗慢性心力衰竭的体外及体内研究提供了实验基础.     

重组腺相关病毒载体的免疫性研究及其解决策略

重组腺相关病毒载体（rAAV）作为一种高靶向性和良好安全性的病毒载体,在基因治疗的临床前和临床研究中具有极广泛的应用前景。目前在全球范围内已有71项临床研究在进行之中或已经完成。其中有相当部分在血友病、视网膜疾病、帕金森、囊性纤维化以及癌症等疾病的治疗中都取得了显著疗效。随着临床上的成功,人们对rAAV所引起的免疫性及其应对策略也进行了更为广泛而深入的研究。本文将就rAAV的外壳蛋白以及目的基因产物所引起的免疫性进行综合探讨,并结合我们的部分研究工作从改变外壳蛋白、给药方式以及消除内源性外壳蛋白的表达等方面对消除外壳蛋白所引起的免疫性的一些策略加以详述。     

大鼠核因子-κB反义RNA腺病毒载体的构建及其在血管平滑肌细胞中的表达

目的:构建表达大鼠核因子κB(NF- κB ,p6 5 )反义RNA的重组腺病毒,并测定受染血管平滑肌细胞(VSMCs)中NF -κB基因的表达变化。方法:从自发性高血压大鼠(spontaneouslyhypertensiverat,SHR)的VSMCs中提取总RNA ,经RT PCR方法获得含全部编码序列的NF κB (p6 5 )cDNA片段(16 5 3bp) ,用TA克隆技术插入pMD18 T载体,构建pMD18 T/16 5 3重组质粒,并经酶切和DNA测序鉴定。以该质粒为模板,经PCR获得NF κB(p6 5 ) 3’端2 6 9bp片段,逆向插入pcDNA3.1( )真核表达载体并置于CMV启动子下。由此构建的pcDNA3.1( ) /2 6 9质粒含有NF κB(p6 5 )的2 6 9bp反义RNA完整表达框。随后将该表达框克隆到入门载体pENTR4 ,构建重组质粒pENTR4 /CMV/2 6 9。使用同源重组方式在体外将该表达框重组到腺病毒载体pAd/PL DEST ,最终得到重组腺病毒质粒pAd/CMV/2 6 9。线性化的重组腺病毒质粒转染2 93细胞后产生的重组腺病毒颗粒用于VSMCs的感染。WesternBlot测定感染前后VSMCs中NF- κB(p6 5 )表达的变化情况。结果:构建的pMD18 T/16 5 3重组质粒经过序列测定,证实其包含大鼠NF κB(p6 5 )基因的16 5 3bp片断;pcDNA3.1( ) /2 6 9、pENTR4 /CMV/2 6 9、pAd/CMV/2 6 9等重组质粒经内切酶消化或PCR等方法鉴定无误;线性化的pAd/CMV     

RIP140重组腺病毒的构建及在乳鼠心肌细胞中的表达

目的原代乳鼠心肌细胞为终末期的分化细胞,常规的质粒转染效率低下。受体相互作用蛋白140(receptor-interacting protein 140,RIP140)目的基因长达3.5kb,为研究RIP140蛋白在非增殖型的心肌细胞表达情况,需要寻找一种更好的过表达系统。方法 RIP140基因全长序列克隆入p Ad Track-CMV穿梭载体中,在BJ5183细菌中与腺病毒骨架载体p Ad Easy~(-1)进行同源重组。经酶切及测序验证的阳性重组克隆转染入AD293细胞中进行病毒的包装与扩增。采用TCID50法测定病毒滴度、CCK-8试剂盒分析腺病毒对心肌细胞活力的影响。采用Western blot鉴定RIP140蛋白在心肌细胞中的表达情况。结果测序分析结果表明RIP140的全长序列正确克隆到Ad Easy TM腺病毒系统中。AdRIP140、Ad-GFP的病毒滴度分别为1011.3和1011.7PFU·m L~(-1)。腺病毒感染复数在200以下时,对心肌细胞活力无影响。绿色荧光及Western blot分析显示Ad-RIP140感染心肌细胞12 h,RIP140表达明显增加(P<0.05)。结论成功构建了RIP140全长序列的腺病毒载体,并使其蛋白有效表达于心肌细胞中,这将有助于后续进行RIP140在心肌细胞中的作用的研究。     

重组脂联素腺相关病毒载体对大鼠滑膜细胞TNF-a、IL-6和BAFF mRNA表达的影响

目的 研究重组脂联素(AD)腺相关病毒载体对大鼠滑膜成纤维细胞(SFLs)肿瘤坏死因子a(TNF-a)、白细胞介素6(IL-6)和B细胞激活因子(BAFF)mRNA表达的影响.方法 采用胶原酶消化法制备大鼠SFLs.携带AD基因的腺相关病毒按不同转染复数(MOI)转染大鼠SFLs,于第7天采用RT-PCR方法检测细胞因子AD、TNF-a、IL-6、BAFF mRNA表达.结果 成功培养出大鼠SFLs,转染后的SFLs AD mRNA表达随MOI升高而增加;在MOI=5×10<'5> v.g./cell时,TNF-a、IL-6、BAFF mRNA表达最高.结论 大鼠SFLs中高表达AD能够促进TNF-a、IL-6和BAFFmRNA表达,提示AD参与炎性反应和B细胞活化,在类风湿关节炎的慢性炎症过程中发挥重要作用.     

携带人生存素基因短发夹RNA表达载体重组腺病毒的构建及其生物学作用

目的构建携带人生存素(survivin)基因短发夹RNA(shRNA)表达载体的重组腺病毒。方法构建并筛选重组质粒pShuttles-urvivin,将其与腺病毒骨架质粒共转染HEK-293细胞,经同源重组产生腺病毒;PCR鉴定并测定病毒滴度;β-半乳糖染色检测病毒对人乳腺癌细胞MCF-7的感染效率;流式细胞术、RT-PCR和W estern免疫印迹法检测其生物学功能。结果经PCR鉴定重组腺病毒构建成功;48 h后感染率达75%以上;腺病毒感染MCF-7细胞后48 h,生存素mRNA和蛋白的表达均受到抑制;腺病毒感染MCF-7细胞后,细胞分裂受阻于G2/M期,在48和72 h有凋亡峰出现。结论成功构建人生存素基因shRNA表达载体的重组腺病毒。     

pU-VEGF-siRNA真核表达载体的构建及鉴定

目的构建针对血管内皮生长因子的发卡样siRNA真核表达载体pU-VEGF-siRNA,为进一步研究其对动脉粥样硬化的作用奠定基础。方法人工合成一对互补并编码相应短发夹状VEGF-siRNA的寡核苷酸链,将其插入到pSilencerTM2.1-U6 neo载体中,经测序鉴定所构建的重组载体是否正确。结果测序示pU-VEGF-siRNA序列正确。结论测序结果表明pU-VEGF-siRNA真核表达载体构建成功。     

DSB修复基因hKu70反义RNA真核表达载体构建

目的 构建人DNA双链断裂（DSB）修复基因hKu70反义RNA真核表达载体pEGFP-C1-K，为以后的hKu70基因功能和毒理学研究提供实验材料。方法 提取人胚肺成纤维细胞（HLF）总RNA，逆转录酶-多聚酶链式反应（RT-PCR）扩增hKu70基因cDNA保守序列，经与pGEM-T载体连接，筛选，克隆，抽提质粒和双酶切后，将纯化的hKu70基因cDNA保守序列反向插入绿色荧光蛋白表达载体pEGFP-C1中，筛选，克隆，抽提质粒，从而构建hKu70基因反义RNA真核表达载体pEGFP-C1-K。结果 经RT-PCR获得467bp含限制性内切酶位点的DNA片段，T载体克隆后经双酶切，测序，确定该片段为hKu70基因cDNA，进而构建反义RNA真核表达载体pEGFP-C1-K，并双酶切，测序确证。结论 成功构建hKu70基因反义RNA真核表达载体pEGFP-C1-K，为建立该基因低表达细胞株，DNA双链断裂修复缺陷和有关毒理学研究提供工具。     

Gene transfer into the CNS using recombinant adeno-associated virus: analysis of vector DNA forms resulting in sustained expression

Recombinant adeno-associated virus (rAAV) vectors have shown significant promise as vehicles for in vivo gene transfer, particularly for transduction of organs composed primarily of non-dividing cells (i.e., muscle, CNS, and liver). However, the mechanistic basis for this desirable property remains unclear. To investigate the fate of rAAV genomes in mouse brain, we stereotactically injected an rAAV vector carrying the E. coli lacZ gene into the caudate of BALB/c mice and demonstrate efficient transduction of mouse brain cells that possess cellular morphology consistent with post-mitotic neurons. We observed a significant increase in beta-galactosidase expression from 5 to 56 days after injection that paralleled the disappearance of single-stranded DNA input genomes. Analysis of in vivo viral DNA forms over time out to 5 months after inoculation revealed that rAAV genomes associated with high molecular weight mouse chromosomal DNA by 14 days after injection and persisted for the length of this study. The pattern of Southern hybridization was consistent with random viral integration in predominantly head-to-tail concatameric arrays. Importantly, we also documented an additional DNA species that appears to be a monomeric episomal circular form based on nuclease sensitivity assays. These data are the first to document the existence of multiple vector DNA forms present within the adult murine brain following direct rAAV inoculation and therefore, provide insight into the molecular events that ultimately result in long-term rAAV mediated transgene expression.     

大鼠CRH基因RNA干扰慢病毒载体的构建与鉴定

目的：构建针对大鼠促肾上腺皮质激素释放激素（CRH）基因的RNA干扰（RNAi）慢病毒表达载体并进行鉴定。方法针对CRH基因设计4个RNAi靶点并分别构建入慢病毒骨架载体,测序鉴定。过表达质粒和RNAi质粒共转染293T工具细胞后,用Western blot进行外源筛靶以确定有效靶序列。有效RNAi病毒质粒与辅助质粒通过Lipofectamine 2000共转染293T细胞,培养48 h后,收集细胞培养上清液,将其浓缩后用孔稀释法测定病毒滴度。结果 Western blot外源筛靶显示3个靶点能有效敲减目的基因的表达。测定浓缩病毒悬液的滴度为1．5×109 T U／ml。结论成功构建了CRH基因的RNAi慢病毒载体,可用于CRH在应激反应中作用的研究。     

蚓激酶重组逆转录病毒载体pLN-BCP-LK~R的构建及其在山羊乳腺组织中的表达

目的在山羊奶中表达蚓激酶。方法将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中获得了蚓激酶重组逆转录病毒载体,转染泌乳期奶山羊乳腺小叶并获得暂时表达。利用脂质体转染PA317包装细胞,G418进行筛选得到阳性细胞克隆,克隆扩大培养后病毒上清感染妊娠期奶山羊乳腺小叶组织,山羊产后采奶纤维蛋白平板溶圈法检测蚓激酶活性。结果产后奶样经纤维蛋白平板溶圈法检测羊奶具有明显纤溶活性。结论蚓激酶基因已经整合到奶山羊乳腺组织并能实现相对稳定的表达。     

Construction and identification of lentiviral RNA interference vector of rat leptin receptor gene

Leptin resistance is a main mechanism of acquired childhood obesity, and the suppression of long form of leptin receptor (OBRb) gene expression in dietinduced obese rats indicates that the down-regulation of OBRb gene expression plays a pivotal role in the mechanism of leptin resistance. The aim of the present study was to construct the lentiviral RNA interference (RNAi) vector of rat OBRb gene and evaluate the effects of siRNA on silencing OBRb gene expression. The target sequence of siRNA-OBRb was designed, and the complementary DNA containing both sense and antisense oligonucleotides was synthesized. After phosphorylation and annealing, these double-stranded DNA was cloned to pRNA-lentivector-VGFP to construct pRNA-Lenti-OBRb-VGFP recombinants with U6-containing promoter, target sequence and Poly III terminator. Then, the products were con.rmed by electrophoresis and sequencing analysis, and the effects of RNAi on reducing gene expression were further con.rmed by real-time polymerase chain reaction in transfected rat glioma cells expressing OBRb. The target sequence of siRNA-OBRb was successfully cloned to pRNA-lentivector-VGFP, and the RNAi protocol speci.cally reduced the expression of OBRb mRNA by approximately 80% compared with controls in transfected rat glioma cells. The successful construction of rat lentivirus vectors expressing OBRb-specific shRNA may be useful for further investigation in vivo.     

携带反义MDR1基因的逆转录病毒载体的构建和表达

目的　建立逆转录病毒介导的反义多药耐药MDR1基因转移体系 ,探讨反义RNA封闭白血病耐药细胞中MDR1基因表达的能力。方法　逆转录 聚合酶链反应 (RT PCR)法从白血病耐药细胞株HL 6 0 /VCRmRNA中扩增出含有启始密码子ATG的 5 2 4bp的MDR1基因cDNA片段 ,反向插入逆转录病毒载体 pLXSN ,通过脂质体转和乒乓感染单、双噬型包装细胞GP +E86和GP +envAM 12 ,以双嗜型病毒上清感染白血病耐药细胞K5 6 2 /MDR ,半定量RT PCR和流式细胞术(FCM)分析MDR1基因转录和P 糖蛋白 (P gp)的表达。 结果　酶切、PCR和DNA测序证实反义MDR1基因重组逆转录病毒载体 pLASSN构建正确 ;双嗜型病毒生产细胞的滴度为 1 3× 10 5CFU/ml,巢式PCR和补救分析均未检测到辅助病毒存在 ;PCR和RT PCR分析证实病毒生产细胞和反义修饰的耐药细胞K5 6 2MDR/LASSN中均有反义MDR1基因整合和MDR1基因反义RNA的转录 ;FCM分析K5 6 2MDR/LASSN细胞中P gp表达强度和表达率比对照细胞均有明显下降。 结论　逆转录病毒介导的反义基因转移系统安全有效 ,可用于肿瘤耐药逆转的研究     

Recombinant adeno-associated virus: clinical application and development as a gene-therapy vector

Gene therapy is gaining momentum as a method of treating human disease. Initially conceived as a strategy to complement defective genes in monogenic disorders, the scope of gene therapy has expanded to encompass a variety of applications. Likewise, the molecular tools for gene delivery have evolved and diversified to meet these various therapeutic needs. Recombinant adeno-associated virus (rAAV) has made significant strides toward clinical application with an excellent safety profile and successes in several clinical trials. This review covers the basic biology of rAAV as a gene therapy vector as well as its advantages compared with other methods of gene delivery. The status of clinical trials utilizing rAAV is also discussed in detail. In conclusion, methods of engineering the vector to overcome challenges identified from these trials are covered, with emphasis on modification of the viral capsid to increase the tissue/cell-specific targeting and transduction efficiency.     

hMSH2反义核糖核酸表达质粒的构建

利用逆转录-聚合酶链反应（RT-PCR）, 特异地扩增HeLa细胞中hMSH2 cDNA的最保守区域, 并将其克隆到TA载体, 从重组质粒两端进行序列分析, 证实为目的片段. 将该目的片段反向克隆到哺乳动物表达载体pREP9的 BamHⅠ和KpnⅠ位点之间, 筛选后得pREP9-hMSH2反义表达重组质粒. 用磷酸钙-DNA共沉淀法将重组质粒DNA及载体DNA分别导入HeLa细胞, 经G418筛选, 扩大培养. 凝胶阻滞实验证实转染有pREP9-hMSH2重组质粒的HeLa-MSH2细胞抽提物中G·T和A·C错配结合蛋白质表达明显降低, 为研究hMSH2基因功能提供了一有效细胞系.     

靶向大鼠血管紧张素转换酶基因的短发夹RNA重组腺病毒载体的构建及鉴定

目的利用AdMax腺病毒载体系统构建大鼠血管紧张素转换酶-短发夹RNA(ACE-shRNA)腺病毒载体并在人胚胎肾-293细胞中扩增制备重组病毒。方法自先期构建的pGenesil-1-ACE-shRNA真核表达载体中用反转录聚合酶链反应(RT-PCR)法扩增出ACE-shRNA片段,并克隆进入穿梭质粒pDC316中,将构建好的穿梭质粒pDC316-ACE-shRNA载体和骨架病毒pBHGlox-E1,3Cre共转染293细胞,包装成重组的病毒颗粒,荧光显微镜观察绿色荧光表达。结果经限制性内切酶,聚合酶链反应(PCR)检测和绿色荧光蛋白(FGFP)表达证实成功地构建了携带ACE-shRNA的重组腺病毒载体并制备出高滴度重组病毒。结论成功地构建了携带ACE-shRNA片段的重组腺病毒载体,为进一步利用基因沉默技术抗高血压的研究奠定了基础。     

靶向大鼠血管平滑肌细胞STAT3的慢病毒干扰载体的构建及鉴定

目的构建并鉴定靶向大鼠血管平滑肌细胞信号传导子及转录激活子3(STAT3)基因的慢病毒干扰载体。方法针对STAT3基因的不同部位设计4对短发卡RNA(shRNA)的寡核苷酸片段,克隆到慢病毒干扰载体PLKO.1中,构建shRNA慢病毒干扰载体PLKO.1-STAT3-shRNA-1、2、3、4,并感染大鼠胸主动脉血管平滑肌细胞。Western blot法检测并筛选最佳抑制效率的shRNA干扰载体。结果 Western blot法证实PLKO.1-STAT3-shRNA-1、2、3、4均能抑制细胞内STAT3基因的表达,以PLKO.1-STAT3-S1最为明显。用PLKO.1-STAT3-S1转染血管平滑肌细胞后可以促进细胞凋亡。结论成功构建了靶向STAT3基因的shRNA慢病毒干扰载体。     

大鼠HSP27基因RNA干扰慢病毒载体的构建与鉴定

目的构建大鼠的热休克蛋白27(heat shock protein27,HSP27)RNA干扰慢病毒载体。方法设计并合成3对互补针对大鼠HSP27 mRNA的oligoDNA片段。磷酸化和退火后,分别克隆入pSUPER-basic质粒载体,获重组的pSU-PER-HSP27-oligoDNA。将其中表达shRNA的结构酶切插入慢病毒转移质粒pNL-IRES2-EGFP,产生pNL-HSP27-IRES2-EGFP,在293T细胞中与VSVG、pHelper包装产生慢病毒。48 h后收集上清液,离心过滤后进行病毒滴度测定。用构建正确的慢病毒质粒载体感染血管平滑肌细胞(vascularsmooth muscle cells,VSMCs),检测干扰效果。结果酶切和测序两种方法结果证明3种质粒载体的插入序列完全正确,慢病毒载体构建成功并获得相应的慢病毒,病毒悬液的滴度为3.12×109fu.L-1。重组慢病毒质粒pNL-HSP27-IRES2-EGFP-1的RNA干扰效率最高,为0.771。结论成功构建靶向大鼠HSP27基因RNAi慢病毒载体。为进一步研究HSP27功能和用慢病毒进行基因治疗奠定基础。     

Treatment of atherosclerosis by transplantation of bone endothelial progenitor cells over-expressed paraoxonase-1 gene by recombinant adeno-associated virus in rat

Endothelial dysfunction/loss is a key event in the development of vascular diseases, including native atherosclerosis (AS). Recent studies have shown that endothelial progenitor cells (EPCs) have the ability to repair endothelial cells that have been lost or damaged following AS. As a result, the therapy of transplanting EPCs is a promising option for the treatment of AS. However, the therapeutic effect on AS with only EPCs transplantation has not been satisfactory. The upregulation of those genes, which prevent the progress of AS in EPCs, is a novel therapeutic strategy for AS. Because it can reduce macrophage foam cell formation and protect endothelial cells from the oxidation of low-density lipoprotein (ox-LDL), paraoxonase-1 (PON1) gene is a candidate for gene therapy in AS. In this study, a recombinant adeno-associated virus (rAAV) vector carrying the human paraoxonase-1 (hPON1) gene (rAAV-PON1) was constructed, and endothelial progenitor cells (EPCs) transfected with rAAV-PON1 were transplanted into the atherosclerosis model of Sprague-Dawley rats (SD rats). The results of doppler ultrasound and histological analysis showed that the group transplanted with the hPON1 gene-transfected EPCs was superior to the group transplanted only with the EPCs and was also better than the group with hPON1 gene injection alone. The results indicated that rAAV-mediated hPON1 gene-transfected EPCs is a potentially valuable new tool in the treatment of atherosclerosis.     

Recombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector

Recombinant adeno-associated virus (AAV)-based vectors capable of expressing therapeutic gene products in vivo have shown significant promise for human gene therapy. One challenge facing the field is the development of vector formulations to achieve optimal vector safety, stability and efficacy. Formulation challenges for AAV vectors can be divided into those relating to maintaining vector activity during purification and storage, and those relating to efficient target tissue transduction in vivo. AAV vectors are potentially susceptible to loss of activity through aggregation, proteolysis and oxidation, as well as through non-specific binding to product contact materials used for vector purification and storage. These deleterious changes need to be thoroughly characterized, and the conditions and excipients to prevent them need to be identified. For in vivo administration, major vector formulation challenges include optimization of efficiency and specificity of target tissue transduction, and the ability to overcome host immune responses.