鸡贫血病病毒VP_2基因在大肠杆菌中的表达及其特性

将鸡贫血病病毒 (chicken anaem ia virus,CAV) VP2 基因克隆入表达性载体 p GEX- 5 X- 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。     

鸡贫血病病毒VP2基因在大肠杆菌中的表达及其特性

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。     

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

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。     

鸡贫血病毒感染鸡细胞凋亡研究

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。     

凋亡素（apoptin）诱导人白血病细胞的．凋亡

为了观察凋亡素（apoptin)能否诱导白血病细胞发生凋亡，利用脂质体介导将凋亡素基因真核表达质粒转染白血病细胞系。通过琼脂糖胶电泳分析、电子显微镜观察，结果表明，凋亡素能促使白血病细胞发生细胞凋亡。     

鸡传染性贫血病毒VP2基因的克隆和原核表达

从组织病料中提取到的鸡贫血病毒核酸经PCR扩增得到vp2基因,并将其克隆到表达载体pET32a上,重组菌经IPTG诱导,SDS-PAGE分析,结果VP2基因在大肠杆菌中成功表达。以表达产物免疫小鼠4次,制备了抗CAVVP2的多克隆抗体。通过对CAV感染的MSB1细胞做间接免疫荧光试验(IFA),结果为阳性。这表明表达产物保留了CAV相关的抗原性。为进一步研究CIA临床诊断奠定了基础。     

我国部分地区鸡贫血病毒全基因组核苷酸序列的比较与分析

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.     

鸡贫血病毒衣壳蛋白VP1基因的克隆、原核表达和纯化

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.     

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

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。     

三肽囊素对环磷酰胺诱导的免疫器官细胞凋亡的影响

研究了三肽囊素对环磷酰胺诱导的鸡免疫抑制模型中免疫器官细胞凋亡的影响。将180只1日龄粤黄鸡随机分成环磷酰胺组、正常对照组、三肽囊素组以及三肽囊素 环磷酰胺组。利用末端脱氧核苷酸转移酶介导的dTUP缺口末端标记法(TUNEL)，对不同组鸡的免疫器官细胞凋亡进行计数。结果发现，环磷酰胺组细胞凋亡数比对照组明显增多(P＜0．05)，而三肽囊素 环磷酰胺组细胞凋亡数显著少于环磷酰胺组(P＜O．05)；三肽囊素组的细胞凋亡数比环磷酰胺组少，且差异显著(P＜0．05)。由此可知，三肽囊素对环磷酰胺所诱导的细胞凋亡具有抑制作用。     

Chicken anemia virus induced apoptosis: underlying molecular mechanisms

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.     

凋亡素基因的克隆

为了探索应用凋亡素杀死肿瘤细胞的可能性,用ＰＣＲ方法扩增了鸡贫血病毒（ｃｈｉｃｋｅｎａｎｅｍｉａｖｉｒｕｓ,ＣＡＶ）的ＶＰ３基因（凋亡素）片段,然后将该片段重组于ｐＵＣ１９载体,并进行了限制性内切酶鉴定与序列分析,证实该片段与鸡贫血病毒Ｃｕｘ－１株的ＶＰ３ＤＮＡ序列一致,该ＤＮＡ全长３６３ｂｐ,编码１２１个氨基酸。     

Cloning and expression of chicken anemia virus VP3 protein in Escherichia coli

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.     

基因分析实现CAV VP1基因在大肠杆菌中的高效表达

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。     

凋亡素诱导的肿瘤细胞凋亡

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。     

Effect of the VP3 gene of chicken anemia virus on canine mammary tumor cells

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.     

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

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.     

抗CDV、CAV卵黄抗体的制备

本研究用CDV、CAV分别免疫产蛋鸡，收集鸡蛋，用海藻酸钠—硫酸铵法提取卵黄抗体(IgY)，并对提取的IgY采用了SDS-PAGE和低压层析检测纯度，结果表明用该法提取的IgY纯度较高。用间接BUSA测定提取的IgY活性，结果证明提取的抗CAV IgY的活性较高，而抗CDV IgY的活性损失较大。