鸡减蛋综合征病毒DNA的基因文库构建和酶切位点分析

腺病毒载体已经成为基因工程疫苗及癌症、遗传病基因治疗的重要工具。腺病毒可分为哺乳动物腺病毒和禽腺病毒,禽腺病毒包括20多个血清型,分别归属于三个群:Ⅰ群为传统的禽腺病毒(FAV),Ⅱ群包括火鸡出血性肠炎病毒(HEV)等,Ⅲ群禽腺病毒为减蛋综合征病毒(EDSV)。这些病毒广泛地存在于多种禽类的呼吸道、消化道,大多呈显性或不显性感染。以禽类腺病毒为载体表达禽类重要病原的保护性抗原基因,构建多价(联)活载体基因工程疫苗,有其重要特点:腺病毒的某些毒株可以在禽体     

抑癌基因PTEN在线粒体上的定位促进A431细胞的凋亡

为研究抑癌基因PTEN在线粒体上的定位对细胞凋亡的影响,构建N端融合有细胞色素C氧化酶亚基七(CoxⅦ)的PTEN腺病毒重组体(Mito-PTEN)。将CoxⅦ-PTEN片段克隆至腺病毒穿梭载体pAdTrack-CMV中,PmeⅠ线性化穿梭质粒,并与腺病毒基因组载体pAdeasy-1共转化大肠杆菌菌株BJ5183,鉴定重组体,并将阳性重组体转化至大肠杆菌菌株DH5α中进行扩增;经PacⅠ酶切后的重组体通过脂质体方法转染用于腺病毒包装的细胞HEK-293A,包装重组腺病毒,收集病毒液,反复扩增,进行病毒滴度的测定;通过绿色荧光蛋白(GFP)标签检测转染及感染效率;用Mito-PTEN腺病毒感染人表皮鳞状癌细胞A431,以流式细胞分析仪检测细胞凋亡情况。成功构建了Mito-PTEN的腺病毒重组体,并进行了该重组体的病毒包装,滴度为107pfu/mL,通过免疫印迹检测目的蛋白,并发现Mito-PTEN可以促进A431细胞的凋亡。Mito-PTEN的成功构建以及它对A431细胞凋亡的促进作用为研究PTEN在线粒体上的功能以及可能的在肿瘤治疗方面的应用提供了理论依据。     

腺病毒感染胞核特殊型类包涵体的电镜观察

一株分离的4型腺病毒在感染细胞核内病毒晶体中出现特殊型类包涵体。该包涵体呈长空芯管样,长度可达1.5μ,外径约为70nm,内径约20nm,可视为与病毒衣壳发育形成有关的形态结构。     

腺病毒及腺病毒相关病毒载体与基因治疗

腺病毒及腺病毒相关病毒能感染增殖与静止的细胞,宿主范围广,其稳定,安全滴度高,转染效率高,腺病毒载体能承载较大的外源基因,腺病毒相关病毒载体能定向整合入靶细胞基因组内等特点,使腺病毒及腺病毒相关病毒载体成为体内用于基因治疗的理想运载系统。     

猪繁殖与呼吸综合征病毒GP5蛋白重组腺病毒的构建与免疫原性测定

应用RT-PCR方法扩增出猪繁殖与呼吸综合征病毒国内分离株S1毒株的GP5基因序列,然后通过KpnI和XhoI酶切位点把该基因克隆入经过同样双酶切的穿梭载体pShuttle-CMV中。重组穿梭载体经过酶切和PCR鉴定后进行测序,证明所克隆入的基因以正确的阅读框插入。获得的重组穿梭载体经线性化后与腺病毒骨架载体共转化大肠杆菌BJ5183菌株。纯化的重组腺病毒质粒经酶切线性化后转染293细胞获得重组腺病毒。重组腺病毒经纯化后进行RT-PCR和间接免疫荧光鉴定,证明了用PRRSVGP5蛋白基因所构建的重组腺病毒成功的表达了GP5蛋白。猪体免疫试验后收集血清进行中和实验证明所构建的重组腺病毒在猪体内能够诱导产生中和抗体,因此我们所构建的重组腺病毒可以作为PRRSV基因工程疫苗研究候选病毒株。     

与CIK细胞结合的肿瘤特异性增殖腺病毒KGHV400的构建

为了构建能与CIK细胞(Cytokine induced killer cell,CIK)结合的肿瘤特异性增殖腺病毒,我们用化学合成的35型腺病毒(Ad35)纤毛基因替换5型腺病毒(Ad5)骨架质粒的纤毛基因,获得5/35嵌合型腺病毒骨架质粒,然后与携带肿瘤特异性启动子hTERT(端粒酶逆转录酶基因启动子)和HRE(缺氧反应元件启动子)的穿梭质粒在HEK293细胞中同源重组,获得能与CIK细胞结合的肿瘤特异性增殖腺病毒KGHV400。分离人外周血单个核细胞,诱导培养成CIK细胞,与KGHV400共培养获得荷载KGHV400的CIK细胞。建立乳腺癌裸鼠移植瘤模型,经尾静脉注射荷载KGHV400的CIK细胞,在注射后第1d、2d、3d、5d、7d、14d解剖实验鼠,用免疫组化技术检测腺病毒六邻体蛋白在移植瘤、心、肝、脾、肾组织的表达。结果显示,构建的重组腺病毒KGHV400能高效率感染CIK细胞;荷瘤鼠尾静脉注射荷载KGHV400的CIK细胞后第1d,肿瘤细胞中即有腺病毒六邻体蛋白表达,之后表达逐渐增多,第14d仍有表达;脾脏在第1~14d检测到少量淋巴细胞中存在腺病毒六邻体蛋白;心、肝、肾组织未检测到该蛋白。对照组(尾静脉注射KGHV400)在注射后第1d、2d、3d、5d,肝、脾、肾组织中检测到存在腺病毒六邻体蛋白,但第7d后为转阴性;肿瘤细胞和心肌细胞未检测到腺病毒六邻体蛋白。我们的研究结果表明,重组腺病毒KGHV400是一种有用的肿瘤基因治疗载体。     

PRKAG2基因新突变体重组腺病毒载体的构建及其在心肌细胞中的表达

目的:构建携带人全长PRKAG2基因新突变体(PRKAG2 G100S)及增强型绿色荧光蛋白报告基因(EGFP)的重组腺病毒载体,并将其在乳鼠心肌细胞中表达.方法:利用Invitrogen的GatewayTM技术,通过重叠PCR技术将目的基因突变体PRKAG2G100S-IRES-EGFP及野生型PRKAG2-IRES-EGFP定向克隆至入门载体pDONR221上,然后与腺病毒载体pAd/CMV/V5-DEST重组反应,形成包含目的基因及EGFP的腺病毒表达克隆.经筛选阳性表达克隆并测序验证后,Pacl酶切、包装、扩增、纯化获得携带EGFP及PRKAG2基因的重组体腺病毒.病毒PCR鉴定并将其感染原代培养的SD乳鼠心肌细胞.用Western blot技术检测PRKAG2蛋白的表达.结果:成功构建了携带人PRKAG2 G100S及EGFP基因的重组腺病毒,病毒的滴度为8.4×108 pfu/ml.荧光显微镜下可见Ad-PRKAG2 G100S重组体腺病毒感染后的乳鼠心肌细胞表达EGFP而发出绿色荧光,PCR证实重组腺病毒载体构建成功,Western blot证明PRKAG2蛋白在乳鼠心肌细胞中过表达.结论:成功构建了携带EGFP基因的Ad-PRKAG2 G100S重组腺病毒载体并将其在乳鼠心肌细胞中正确表达,为今后突变体PRKAG2基因的功能研究奠定了基础.     

腺病毒感染及胞内运输途径的研究进展

目前关于腺病毒感染及胞内运输的分子机制研究主要来源于C亚群腺病毒在肿瘤细胞系中的研究结果。腺病毒对靶细胞的感染及胞内运输大致分为几步:病毒与细胞表面受体的特异结合,胞吞介导的病毒内化,病毒逃脱胞内体进入细胞质,病毒沿着微管运输至核孔,病毒基因组入核。病毒胞内运输效率极高,感染后1 h,80%以上的病毒基因组被送至核内。但是腺病毒胞内的运输方式会因以下几个因素变化而产生差异:靶细胞类型,细胞生理状态,病毒血清型。文中对腺病毒感染靶细胞及胞内运输的已有分子机制进行综述,为临床基因治疗用途的病毒载体研发提供思路。     

腺病毒相关病毒携带低密度脂蛋白受体基因对实验性高胆固醇血症的治疗作用

低密度脂蛋白受体 (LDL- R)对于血清胆固醇的清除和预防动脉粥样硬化的发生起重要作用 .用构建了含有LDL- R基因的重组腺病毒相关病毒 ,通过腺病毒包被蛋白脂质体 (Adenosome) ,转移至食饵性高脂血症家兔的肝脏内 ,证明可以降低血浆胆固醇水平 ,为高胆固醇血症的基因治疗提供一条新途径     

腺病毒介导犬干扰素-γ基因的表达及其体外抗犬细小病毒的作用

[目的]构建含犬干扰素-γ(c IFN-γ)基因的重组腺病毒,并在培养的犬肾细胞MDCK中分析其抗犬细小病毒的活性.[方法]首先将cIFN-γcNDA基因克隆到腺病毒穿梭质粒中,构建成含cIFN-γ基因的腺病毒穿梭质粒pShuttle3-cIFN-γ.利用特异的酶切位点,通过直接连接法将cIFN-γ表达盒插入到腺病毒基因组质粒pAdeno-X中,构建成含cIFN-γ基因的腺病毒基因组质粒pAd-cIFN-γ.pAd-cIFN-γ质粒经酶切线性化后转染人胚胎肾细胞HEK293T,在细胞中拯救出含有cIFN-γ基因的复制缺陷型重组腺病毒.然后用该重组腺病毒处理(感染)培养的犬肾细胞MDCK,再用犬细小病毒感染重组腺病毒处理的细胞,分析重组腺病毒在体外抗犬细小病毒的活性.[结果]通过连接法构建了含cIFN-γ基因的重组腺病毒,构建的重组腺病毒能够介导cIFN-γ在MDCK细胞中进行分泌表达.用含cIFN-γ基因的重组腺病毒处理MDCK细胞,可明显地抑制犬细小病毒在细胞中的增殖,表明构建的重组腺病毒具有明显的抗犬细小病毒的活性.[结论]构建了含cIFN-γ基因的重组腺病毒,并证明该重组病毒在体外具有明显的抗犬细小病毒的活性.     

Gene therapy using AAV

AAV (adeno-associated virus) vectors are considered to be promising gene-delivery vehicles for gene therapy, because they are derived from non-pathogenic virus, efficiently transduce non-dividing cells, and cause long-term gene expression. Appropriate AAV serotypes are utilized depending on the type of target cells. Among various neurological disorders, Parkinson's disease (PD) is one of the most promising candidates of gene therapy. PD is a progressive neurodegenerative disorder that predominantly affects dopaminergic neurons in the substantia nigra. One of the major approaches to gene therapy of PD is the intrastriatal expression of dopamine (DA)-synthesizing enzyme genes. As for the initial step of clinical application, AAV vector-mediated AADC (aromatic L-amino acid decarboxylase; the enzyme converting L-DOPA to DA) gene transfer in combination with oral administration of L-DOPA would be appropriate, since DA production can be regulated by adjusting the dose of L-DOPA. Second, intramuscular injection of AAV vectors is appropriate to protein-supplement gene therapy. Monogenic diseases such as hemophilia and Fabry disease are suitable candidates. Regarding cancer gene therapy, AAV vectors may be utilized to inhibit tumor angiogenesis, metastasis, and invasion. When long-term transgene expression in stem cells is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of AAV would be particularly valuable.     

Gene therapy for Parkinson's disease

Gene therapy is a potentially powerful approach to the treatment of neurological diseases. The discovery of neurotrophic factors inhibiting neurodegenerative processes and neurotransmitter-synthesizing enzymes provides the basis for current gene therapy strategies for Parkinson's disease. Genes can be transferred by viral or nonviral vectors. Of the various possible vectors, recombinant retroviruses are the most efficient for genetic modification of cells in vitro that can thereafter be used for transplantation (ex vivo gene therapy approach). Recently, in vivo gene transfer to the brain has been developed using adenovirus vectors. One of the advantages of recombinant adenovirus is that it can transduced both quiescent and actively dividing cells, thereby allowing both direct in vivo gene transfer and ex vivo gene transfer to neural cells. Probably because the brain is partially protected from the immune system, the expression of adenoviral vectors persists for several months with little inflammation. Novel therapeutic tools, such as vectors for gene therapy have to be evaluated in terms of efficacy and safety for future clinical trials. These vectors still need to be improved to allow long-term and possibly regulatable expression of the transgene.     

Adeno-associated virus vectors for gene transfer to the brain

Gene therapy is a novel method under investigation for the treatment of neurological disorders. Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system. Adeno-associated virus (AAV) is a potentially useful gene transfer vehicle for neurologic gene therapies. The advantages of AAV vector include the lack of any associated disease with a wild-type virus, the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for neurological disorder by using AAV vector has an increasing impact on gene therapy research. This article describes methods that can be used to generate rodent and nonhuman primate models for testing treatment strategies linked to pathophysiological events in the ischemic brain and neurodegenerative disorders such as Parkinson's disease.     

The evolution of heart gene delivery vectors

Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer.     

Gene therapy using herpes simplex virus-based vectors

Gene therapy involves the use of specific genes to treat human diseases and is thus critically dependent on efficient gene delivery systems. Although a variety of systems for such gene delivery are under development, HSV has unique advantages in terms of its large genome size and for gene delivery in the nervous system because of its ability to enter a latent state in neuronal cells. Considerable progress has been made in the effective disablement of this virus whilst retaining its ability to deliver genes and in producing long term expression of the foreign gene. Although much remains to be achieved in the further disablement of the virus and its testing in rodent and primate models of human diseases, it is likely that these viruses may ultimately be of use in human gene therapy procedures particularly for otherwise intractable neurological diseases.     

Gene therapy for Parkinson's disease

Parkinson's disease (PD) is a debilitating neurodegenerative disorder arising from loss of dopaminergic neurons in the substantia nigra pars compacta and subsequent depletion of striatal dopamine levels, which results in distressing motor symptoms. The current standard pharmacological treatment for PD is direct replacement of dopamine by treatment with its precursor, levodopa (L-dopa). However, this does not significantly alter disease progression and might contribute to the ongoing pathology. Several features of PD make this disease one of the most promising targets for clinical gene therapy of any neurological disease. The confinement of the major pathology to a compact, localised neuronal population and the anatomy of the basal ganglia circuitry mean that global gene transfer is not required and there are well-defined sites for gene transfer. The multifactorial aetiology of idiopathic PD means that it is unlikely any single gene will cure the disease, and as a result at least three separate gene-transfer strategies are currently being pursued: transfer of genes for enzymes involved in dopamine production; transfer of genes for growth factors involved in dopaminergic cell survival and regeneration; and transfer of genes to reset neuronal circuitry by switching cellular phenotype. The merits of these strategies are discussed here, along with remaining hurdles that might impede transfer of gene therapy technology to the clinic as a treatment for PD.     

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.     

Bronchoalveolar fluid is not a major hindrance to virus-mediated gene therapy in cystic fibrosis

Successfully targeting the airway epithelium is essential for gene therapy of some pulmonary diseases. However, the airway epithelium is resistant to virus-mediated gene transfer with commonly used vectors. Vectors that interact with endogenously expressed receptors on the apical surface significantly increase gene transfer efficiency. However, other endogenous components involved in host immunity may hinder virus-mediated gene transfer. We tested the effect of bronchoalveolar lavage liquid (BAL) from patients with cystic fibrosis (CF), BAL from subjects without CF (non-CF BAL), Pseudomonas aeruginosa-derived proteins, and an array of inflammatory proteins on gene transfer mediated by adeno-associated virus type 5 (AAV5) and adenovirus targeted to an apically expressed glycosylphosphatidylinositol-modified coxsackie-adenovirus receptor. We found that neither CF BAL nor its components had a significant effect on gene transfer to human airway epithelium by these vectors. Non-CF BAL significantly impaired adenovirus-mediated gene transfer. Removal of immunoglobulins in non-CF BAL restored gene transfer efficiency. As virus vectors are improved and mechanisms of humoral immunity are elucidated, barriers to successful gene therapy found in the complex environment of the human lung can be circumvented.