甲病毒作为基因工程载体的研究进展

披盖病毒科的甲病毒用作基因工程载体有许多特殊的优点,在疫苗制备和基因治疗方面有巨大的应用潜力。最近已证明该系统可用于阻断虫媒病毒的传播,从而开辟了一个全新的研究领域。该系统可能特别适用于制备高效而安全的核酸疫苗。     

马铃薯X病毒分子生物学研究进展及其作为表达载体的应用

马铃薯X病毒(Potato virus X,PVX)又称马铃薯潜隐病毒(Potato latent virus)或马铃薯轻花叶病毒(Potato mild mosaic virus),是马铃薯X病毒属(Potexvirus)的模式成员,遍布于全世界马铃薯种植区.受侵染叶片呈花叶症状,田间常与其他病毒混合感染导致马铃薯的毁灭性减产.     

马铃薯X病毒分子生物学研究进展及其作为表达载体的应用

马铃薯X病毒(Potato virus X,PVX)又称马铃薯潜隐病毒(Potato latent virus)或马铃薯轻花叶病毒(Potato mild mosaic virus),是马铃薯X病毒属(Potexvirus)的模式成员,遍布于全世界马铃薯种植区。受侵染叶片呈花叶症状,田间常与其他病毒混合感染导致马铃薯的毁灭性减产。其病毒粒子为易弯曲的杆状颗粒,大小约为     

TT病毒分子生物学研究进展

TT病毒属圆环病毒科,其基因组为单链负股环状DNA,全长的3.8kb,由编码区和非编码区组成,前者至少含有6个公认的开放阅读框架,TT病毒以滚环复制方式主要在肝细胞和造血细胞中复制,其转录产物包括3.0,1.2,1.0kb等3种剪接mRNA,TT病毒的衣壳蛋白由3.0kbmRNA转译,而1.2kb,1.0kbmRNA表达产物为非结构蛋白,对转录过程起调节作用。最近,有研究发现,TT病毒开放阅读框架表达一种可能对肾上皮细胞有损伤作用的类鱼精蛋白。     

牛泡沫病毒（BSV）3026毒株的分离及分子生物学鉴定

从一头牛免疫缺陷病毒（ＢＩＶ）检测阳性３０２６号牛外周血中,分离到一株病毒,即３０２６病毒株。体外细胞增减和反转录分析证明,此病毒是一反转录病毒。可胎牛肺细胞中引起典型的泡沫样病变,形成合胞体,ＰＣＲ扩增和Ｓｏｕｔｈｅｍ杂交显示,此病毒的ＣＤＮＡ和ＰＣＲ产的均可与牛泡沫病毒（ＢＳＶ）阳性对照的ＨｉｒｔＤＮＡ杂交。３‘ＬＴＲ上游一段３３０ｂｐ的ＰＣＲ产物序理分析表明,３０２６毒株与ＢＳＶ阳性对照相比     

腺病毒相关病毒载体的研究进展

实现基因治疗的关键在于目的基因的高效转移并适度表达,这将直接影响其治疗的效率和安全性。因此,探寻理想的基因转移载体显得尤为重要。腺病毒相关病毒（adeno-associated virus,AAV）是一种缺陷型的单链DNA病毒,既可以转染分裂细胞又可以转染非分裂细胞。AAV在宿主体内以定向整合的方式存在,AAV重组体在细胞内能长期稳定地表达,且在体内不引起明显的病理变化,表明AAV作为基因治疗的载体是安全、有效和可行的。     

人类免疫缺陷病毒重组载体疫苗的研究进展

重组载体疫苗是目前人类免疫缺陷病毒疫苗的研究热点,近几年来不仅在病毒载体和细菌载体选用的种类方面有了新的突破,而且对载体疫苗的组合免疫策略和最佳免疫的选用方面已有全新的认识。本文就用于该病毒重组疫苗的病毒载体(包括痘病毒、α病毒、仙台病毒、甲型流感病毒、腺病毒、狂犬病病毒、疱疹性口炎病毒和单纯疱疹病毒载体)以及细菌载体(包括卡介苗、李斯特单胞茵、沙门茵和布氏杆菌载体)等的研究进展进行综述。     

痘苗病毒载体研究进展

制备高活性的真核蛋白,必须使用哺乳动物细胞,痘苗病毒为这一工作提供了简便、实用的工具．痘菌病毒宿主范围广,无致癌性,插入长达20多kb的外源基因后仍有感染性,能够高效表达外源蛋白,有效地加工表达产物．而且,痘菌病毒的表达产物可刺激机体免疫系统产生良好的体液免疫和细胞免疫．利用这些特点构建成的哺乳动物细胞高效表达系统将是基因工程药物和基因工程疫苗的有效工具.     

腺病毒相关病毒载体的研究进展

腺病毒相关病毒（ＡＡＶ）是一种缺陷型的单链ＤＮＡ病毒,它能感染造血干细胞、脑细胞等非分裂细胞是其显特征之一。现有资料ＡＡＶ载体能以特异定向整合的方式存在。ＡＡＶ重组体在细胞内能长期稳定表达,且在体内未引起明显的病理改变,表明ＡＡＶ作为基因治疗的载体是安全、有效和可行的。     

cis-Acting Sequences Required for Simian Foamy Virus Type 1 Vectors

We have constructed a series of vectors based on simian foamy virus type 1 (SFV-1) to define the minimum cis-acting elements required for gene transfer. To characterize these vectors, we inserted the coding sequence of the bacterial lacZ gene linked to the cytomegalovirus immediate-early gene promoter. Introduction of a deletion mutation in the leader region between the 5′ long terminal repeat and the start of the gag gene at position 1659 to 1694 completely abrogated gene transfer by the SFV-1 vector. Deletion of 39 nucleotides from position 1692 to 1731 in the leader region resulted in a significant reduction in the transducing-particle titer. Furthermore, we have identified a second cis-acting element located at the 3′ end of the pol gene between position 6486 and 6975 to be critical for SFV-1 vector transduction. These results identify the two important cis-acting elements required for SFV-1 vector construction, and the finding of a cis-acting element in the pol gene is unique among retroviruses.     

溶瘤病毒载体研究进展

溶瘤病毒(oncolytic virus,OVs)疗法是治疗肿瘤的一种新方法,它可通过直接杀死肿瘤细胞并引起机体的免疫反应发挥作用.然而天然溶瘤病毒有一定的局限性,因此需将各种不同的病毒作为载体,通过基因修饰的方法增强或减弱病毒毒力并导入新的功能性基因,以提高其溶瘤作用.目前可以作为溶瘤病毒载体的有单纯疱疹病毒、腺病毒、牛痘病毒、水泡性口炎病毒、麻疹病毒、腮腺炎病毒、脊髓灰质炎病毒等.这些病毒载体均可通过不同的基因修饰方法,靶向性感染并杀死不同的肿瘤细胞,获得较好的溶瘤作用.本文综述了几种溶瘤病毒载体的基因修饰方法,以及修饰后的溶瘤效果.     

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.     

Characterization of a cis-Acting Sequence in the pol Region Required To Transfer Human Foamy Virus Vectors

To identify cis-acting elements in the foamy virus (FV) RNA pregenome, we developed a transient-vector-production system based on cotransfection of indicator gene-bearing vector and gag-pol and env expression plasmids. Two elements which were critical for vector transfer were found and mapped approximately. The first element was located in the RU5 leader and the 5′ gag region (approximately up to position 650 of the viral RNA). The second element was located in an approximately 2-kb sequence in the 3′ pol region. Although small 5′ and 3′ deletions, as well as internal deletions of the latter element, were tolerated, both elements were found to be absolutely required for vector transfer. The functional characterization of the pol region-located cis-acting element revealed that it is essential for efficient incorporation or the stability of particle-associated virion RNA. Furthermore, virions derived from a vector lacking this sequence were found to be deficient in the cleavage of the Gag protein by the Pol precursor protease. Our results suggest that during the formation of infectious virions, complex interactions between FV Gag and Pol and the viral RNA take place.     

Carrier Cultures of Simian Foamy Virus

The production of cultures of HEp-2 and BHK-21 cells persistently infected with a type 1 simian foamy virus is described. After infection, HEp-2 cells showed no structural changes, whereas BHK-21 cells lost their normal spindle shape and showed mitochondrial damage, and some cells contained many lysosomes. Thin sections also showed that a few BHK-21 cells contained virus particles in low concentration, and infectious virus could be isolated from both the cells and the supernatant fluid. No virus was seen in thin sections of HEp-2 cells, although infectious virus in low titer could be recovered intermittently from lysed cells. Both carrier cultures were immune to challenge with homologous virus and antigen could be detected in over 90% of the cells even after growth for 9 weeks in the presence of virus-neutralizing serum. The distribution of antigen in carrier cultures of both cell types is described and compared with that seen in cytocidal infections.     

双元Ti载体的发展

双元Ti载体是目前植物基因工程中最重要的植物转化载体。近年来双元Ti载体得到迅速的发展,新的载体不断出现。新发展的双元Ti载体结构优化,适用范围广,易于基因克隆操作,同时提高了植物转化的效率,更便于进行植物基因功能的研究。本文介绍了构建高容量载体、多基因载体、定点整合载体所采用的新策略。