共查询到17条相似文献,搜索用时 156 毫秒
1.
2.
鸡贫血病病毒VP2基因在大肠杆菌中的表达及其特性 总被引:2,自引:0,他引:2
将鸡贫血病病毒(chicken anaemia virus,CAV)VP2基因克隆入表达性载体PGEX-5X-3,在大肠杆菌以谷胱甘肽转移酶(GST)融合蛋白的形式获得了表达。以此表达产物免疫小鼠,制备抗CAV VP2的多克隆抗体。通过对CAV感染的MSB1细胞作间接免疫荧光试验(IFA)检测,结果为阳性,这表明表达产物保留了CAV相关的抗原特性,本研究为进一步探索CAV VP2的生物学特性奠定了基础。 相似文献
3.
第46代鸡贫血病病毒细胞凋亡素基因的体外扩增、克隆、序列比较 总被引:6,自引:1,他引:5
通过聚合酶链式反应(PCR)扩增了第46代鸡贫血病病毒(CAV)Cux株的细胞凋亡素(Appoptin)基因,并克隆入pGEX-5X-3质粒载体中,通过限制性内切酶分析、序列测定,验证了克隆的正确性。细胞凋亡素基因全长为363bp,编码121个氨基酸。与Meehan.等发表的CAV Cux株序列相比,第46代细胞适应毒的细胞素基因的第209个核苷酸由C→T,与澳大利亚株,美国株CVA序列分别有5个和2个碱基的差别,并且均匀分布在5'端前210碱基区,而在3'端序列较为保守。与同处欧洲的英国株CAV序列相比,序列较为一致,本研究对所克隆的凋亡素编码蛋白进行了疏水性分析和抗原表位优势分析。 相似文献
4.
鸡贫血病毒感染鸡细胞凋亡研究 总被引:2,自引:0,他引:2
用TUNEL法检测了1 日龄SPF雏鸡感染鸡贫血病毒(CAV)后胸腺和骨髓细胞的凋亡情况。结果,接种CAV 后6、10、20 d,骨髓细胞的凋亡率分别达51% 、52% 、57% ;接种后6 d,胸腺细胞凋亡率为53% ,比对照鸡胸腺和骨髓细胞的凋亡率明显升高( P< 0.01)。用流式细胞仪对胸腺细胞悬液所作的细胞凋亡分析结果与TUNEL法检测结果一致。 相似文献
5.
6.
7.
鸡贫血病毒(Chicken anemia virus,CAV)是目前已知最小的动物病毒之一,为单股环状DNA,基因组长度为2.3 kb.CAV基因组单链有3个部分或完全重叠的开放阅读框(ORF),分别编码核衣壳蛋白VPl、相关蛋白VP2、细胞凋亡因子VP3 3种蛋白.至今发现的所有CAV毒株的抗原性都相同,均属于同一个血清型,但世界各地的CAV分离株之间毒力不同,基因组序列也存在一些差异. 相似文献
8.
根据GenBank中CAV哈尔滨分离株基因序列,设计出针对CAV VP1大片段(608 bp)的引物,利用PCR方法从CAV基因组序列中扩增出VP1基因的大片段(608 bp),按照正确的读码框克隆到原核表达载体pET32a( )上,得到含VP1大片段的pET32a( )重组子,转化大肠埃希菌BL21(DE3)感受态细胞,IPTG诱导重组蛋白表达,经SDS-PAGE和Western blotting检测发现有分子质量约42 ku的融合蛋白表达,与预期分子质量大小一致,通过Ni亲和层析柱纯化出融合蛋白,经Western blotting鉴定,融合蛋白与His单抗能够结合.CAV VP1基因的克隆表达及其融合蛋白的纯化为后续制备多克隆抗体和单克隆抗体提供了良好的抗原来源,也为研究VP1与其他CAV蛋白之间的相互关系奠定了基础. 相似文献
9.
凋亡素基因真核表达载体的构建及在人肿瘤细胞中的表达 总被引:3,自引:1,他引:2
为探讨凋亡素对肿瘤的基因治疗效果,构建了凋亡素基因的真核表达载体。将凋亡素基因重组入 pc D N A3 载体,并通过脂质体介导凋亡素在人喉癌、人肺癌细胞系中表达;通过 R T P C R 在转染细胞中检测到了凋亡素的 m R N A,这为应用凋亡素进行肿瘤的基因治疗奠定了基础。 相似文献
10.
凋亡素诱导的肿瘤细胞凋亡 总被引:2,自引:1,他引:2
细胞凋亡是机体维持自身稳定的一种基本生理机制 ,并贯穿于整个生命活动。细胞凋亡异常可导致一些疾病的发生 ,如肿瘤的发生。同时 ,细胞凋亡的可诱导性也为肿瘤治疗提供了新的思路。凋亡素是来源于鸡贫血病毒的一个小分子蛋白。它能够选择性地诱导肿瘤细胞的凋亡 ,而对正常二倍体的细胞无任何毒副作用 ,并且正常细胞对凋亡素具有耐受性。凋亡素诱导肿瘤细胞凋亡既不需要依赖 p53 ,也不会被bcl-2的过表达所抑制。凋亡素还能诱导人成骨肉瘤细胞多药耐药株 R-OS-73 2的细胞凋亡。这些特点使凋亡素成为一种新型的候选抗肿瘤制剂。 相似文献
11.
Chicken anemia virus induced apoptosis: underlying molecular mechanisms 总被引:23,自引:0,他引:23
Noteborn MH 《Veterinary microbiology》2004,98(2):89-94
In 1990, the chicken anemia virus (CAV) genome was cloned by us and proven to be representative for CAV isolates worldwide. This genome contains unique promoter/enhancer replication elements and genes. Upon infection of its target cells, CAV replicates via a double-stranded (ds) DNA intermediate. From this ds CAV molecule, a single mRNA is transcribed, which encodes for three distinct proteins VP1, VP2, and VP3 or apoptin. Its capsid contains only the VP1 protein. However, for the production of the neutralizing epitope, co-synthesis of VP1 and VP2 are needed. CAV genomes with mutations in the 12 bp insert of the promoter/enhancer region were shown to produce immunogenic functional CAV particles. Mutations in these and other regulatory elements of CAV might also decrease its virus load resulting in a reduced pathogenic effect. CAV causes fatal cytopathogenic effects in e.g. chicken thymocytes via apoptosis. Under in vitro conditions, CAV replicates only in transformed chicken cell lines, which indicates that at least a part of the CAV life-cycle requires transformed-like cellular events. In these transformed cell lines, the synthesis of the apoptin protein alone mimics the CAV-induced apoptosis, whereas the VP2 protein also harbors some apoptotic activity. Extensive studies on apoptin resulted in the characterization of domains essential for its apoptotic activity and nuclear localization, which seems to be related with its ability to induce apoptosis. Therefore, both VP2 and apoptin are of interest in reducing the pathogenicity of CAV infections. A series of biomedical studies on apoptin have been carried out in human cell systems, which are informative about the mechanism of CAV-induced apoptosis in chicken (transformed) cells. Synthesis of apoptin alone induces apoptosis in various human transformed and/or tumorigenic cell lines, but not in normal human diploid cells. A striking difference in the cellular localization of apoptin was observed in human normal diploid cells versus tumor cells. In all tumor cells, apoptin is located mainly in the heterochromatic regions of the nucleus, whereas in normal cells it is present in peri-nuclear structures. Apoptin contains a bipartite nuclear localization signal, and one domain that resemble a nuclear export signal. Elucidation of parts of the apoptin-induced apoptotic pathway revealed unique characteristics: apoptin-induced apoptosis is independent of the tumor suppressor p53. The anti-apoptotic protein Bcl-2 does not inhibit but even accelerates apoptin-induced apoptosis in tumor cells, whereas over expression of Bcl-2 in normal cells has no effect on the apoptin activity. Upstream caspases are not involved, whereas downstream caspase 3 is, but seems not to be essential. A number of novel proteins were shown to interact with apoptin in transformed cells. Future studies of apoptin, VP2 and related cellular proteins in chicken cells will unravel the regulatory aspects of CAV-induced apoptosis. 相似文献
12.
13.
Nogueira-Dantas EO Ferreira AJ Astolfi-Ferreira CS Brentano L 《Comparative immunology, microbiology and infectious diseases》2007,30(3):133-142
Purification of chicken anemia virus (CAV) VP3 protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. CAV particle was obtained from infected liver of chicken and DNA was extracted. The VP3 protein gene was amplified from the extracted DNA by polymerase chain reaction (PCR) and cloned. The recombinant expression construct (pTrc-VP3) was identified by PCR and sequencing analysis. Expression of VP3 protein with a molecular mass of approximately 21kDa was confirmed by Western blotting analysis with CAV-specific antibodies. The in vitro expressed VP3 protein was purified to near homogeneity by elution from the gel, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP3 protein was recognized by CAV antibodies in a Western blotting assay. This finding indicates that recombinant VP3 expressed in the pTrcHis2 vector system can be used as antigen to detect anti-CAV antibodies. 相似文献
14.
通过分子生物学软件DNASTAR对CAV VP1的基因分析发现,CAV VP1基因的5端非抗原区编码蛋白的密码子中存在连续的大肠杆菌稀有密码子。为了在大肠杆菌中高效表达CAV VP1,文章研究扩增了不含5’端稀有密码子的CAV VP1基因,并将其插入原核表达载体pGEX-4T-1,构建了重组表达载体pGEX-VP1,成功进行了高效表达。电泳条带分析表明,融合蛋白的表达量在44%左右,为研究CAV VP1的抗原性提供了丰富的来源。 相似文献
15.
Yi-Yang Lien Chi-Hung Huang Fang-Chun Sun Shyang-Chwen Sheu Tsung-Chi Lu Meng-Shiunn Lee Shu-Chin Hsueh Hsi-Jien Chen Meng-Shiou Lee 《Journal of veterinary science (Suw?n-si, Korea)》2012,13(1):73-79
Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-β-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future. 相似文献
16.
Lee JJ Chen PB Yang SH Cheng CH Chueh LL Pang VF Hsiao M Lin CT 《American journal of veterinary research》2007,68(4):411-422
OBJECTIVE: To investigate the antitumor effect of the chicken anemia virus (CAV) VP3 gene in canine mammary tumor (CMT) cells. SAMPLE POPULATIONS: Established primary canine cell lines that originated from epithelial cells of resected CMTs and nonneoplastic mammary gland epithelial (MGE) cells. PROCEDURES: Expression vectors and lentiviral vectors encoding the VP3 gene from a Taiwan-Ilan isolate of CAV were used to deliver the VP3 gene into CMT cells and nonneoplastic MGE cells. Ectopic gene expression and the pro-apoptotic effect of the VP3 gene on CMT and nonneoplastic MGE cells by either transfection or viral infection were evaluated via immunofluorescence microscopy, western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis. RESULTS: Overexpression of the enhanced green fluorescent protein-VP3 fusion protein was detected predominantly in the nuclei of CMT cells. In contrast, the VP3 protein was localized to the cytoplasm of nonneoplastic MGE cells. Among the fusion protein-expressing CMT cells, most underwent characteristic changes of apoptosis, whereas apoptosis was not detected in fusion protein-expressing, nonneoplastic MGE cells. Induction of apoptosis by VP3 gene overexpression in CMT cells was associated with the caspase-9-, but not the caspase-8-, mediated apoptosis pathway. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that the VP3 gene of the CAV induces apoptosis in malignant CMT cells, but not in nonneoplastic canine MGE cells. On the basis of such tumor cell-specific killing, the VP3 gene may be a promising agent for the treatment of malignant mammary gland tumors in dogs. 相似文献
17.
Minxiu Zhang Xianwen Deng Zhixun Xie Yanfang Zhang Zhiqin Xie Liji Xie Sisi Luo Qing Fan Tingting Zeng Jiaoling Huang Sheng Wang 《Journal of veterinary science (Suw?n-si, Korea)》2022,23(5)
BackgroundChicken anemia virus (CAV) causes chicken infectious anemia, which results in immunosuppression; the virus has spread widely in chicken flocks in China.ObjectivesThe aim of this study was to understand recent CAV genetic evolution in chicken flocks in Guangxi Province, southern China.MethodsIn total, 350 liver samples were collected from eight commercial broiler chicken farms in Guangxi Province in southern China from 2018 to 2020. CAV was detected by conventional PCR, and twenty CAV complete genomes were amplified and used for the phylogenetic analysis and recombination analysis.ResultsThe overall CAV-positive rate was 17.1%. The genetic analysis revealed that 84 CAVs were distributed in groups A, B, C (subgroups C1-C3) and D. In total, 30 of 47 Chinese CAV sequences from 2005-2020 belong to subgroup C3, including 15 CAVs from this study. There were some specific mutation sites among the intergenotypes in the VP1 protein. The amino acids at position 394Q in the VP1 protein of 20 CAV strains were consistent with the characteristics of a highly pathogenic strain. GX1904B was a putative recombinant.ConclusionsSubgroup C3 was the dominant genotype in Guangxi Province from 2018–2020. The 20 CAV strains in this study might be virulent according to the amino acid residue analysis. These data help improve our understanding of the epidemiological trends of CAV in southern China. 相似文献