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2008年23卷3期

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对虾白斑综合症病毒结构蛋白蛋白质组学研究进展

谭艳微, 石正丽

2008, 23(3): 157 doi: 10.1007/s12250-008-2924-0

对虾白斑综合症病毒(White Spot Syndrome Virus,WSSV)是感染对虾和其它水生甲壳类动物并导致全球水产养殖业严重经济损失的水生病毒,是Nimaviridae科、Wispovirus属的唯一成员。WSSV基因组大小约为300kb,其基因组中推测有181个开放阅读框可以编码蛋白质,然而到目前为止大部分这些编码的蛋白质还未被证实。系统性研究WSSV的结构蛋白,病毒发生过程和病毒组装过程对于控制该病毒的流行是十分必要的,因为结构蛋白在病毒的结构组成,感染过程和与宿主受体相互作用起关键作用,找到病毒吸附和侵入宿主起关键作用的结构蛋白对WSSV的有效防治具有重要意义。本篇文章主要综述了WSSV结构蛋白研究进展,主要包括结构蛋白的数量,定位,功能和在病毒感染过程中它们之间的相互作用以及目前研究结构蛋白的主要方法。通过传统的蛋白质组学研究方法结合一维或二维凝胶电泳-质谱和数据库分析已经鉴定出WSSV 39个结构蛋白。随着蛋白质组学研究方法的快速发展,鸟枪法结合同位素标记相对和绝对定量方法的应用进一步确定了WSSV结构蛋白的总量和定位。到目前为止,已经鉴定出WSSV结构蛋白59个,其中44个蛋白的定位已经确认:35个为囊膜蛋白(包括内膜蛋白),9个为核衣壳蛋白。同时本文综述了WSSV感染过程中起主要作用的结构蛋白和它们之间的相互作用。 这些蛋白的确定有助于揭示病毒的组装过程以及确认它们在病毒侵染过程起到的主要作用。

核酸扩增新技术——环介导的等温核酸扩增

方雪恩, 李健, 陈沁

2008, 23(3): 167 doi: 10.1007/s12250-008-2929-8

环介导的等温核酸扩增是新一代的核酸扩增手段。该方法在一种具有链置换的DNA聚合酶的作用下,通过特殊引物的介导,大量合成DNA,同时具有特异性强、灵敏度高和速度快、成本低等优点。本文通过介绍该方法的基本原理、优点、发展和应用情况等阐述该方法目前的研究状况。

Abstract

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

HIV-1 Vif和人APOBEC3G的原核表达、纯化

李岚, 杨怡姝, 李泽琳, 曾毅

2008, 23(3): 173 doi: 10.1007/s12250-008-2909-z

Abstract为了获得HIV-1 Vif和APOBEC3G及其多克隆抗体,采用PCR技术从HIV-1 NL4.3的质粒中扩增vif基因,采用反转录-聚合酶链式反应(RT-PCR)技术以H9细胞的总RNA为模板扩增APOBEC3G基因。将测序鉴定过的基因克隆到原核表达载体pET-32a上,以包涵体的形式在大肠杆菌BL21(DE3)中高效表达,APOBEC3G蛋白及Vif蛋白C端融合6×His标签便于纯化及鉴定。应用酶切技术、SDS-PAGE及Western blotting等方法确保基因片段的正确性及表达蛋白的特异性。纯化后的APOBEC3G蛋白及Vif蛋白分别免疫日本大耳白兔,间接ELISA法测定多克隆抗体滴度,免疫酶法和免疫荧光法检测多克隆抗体的特异性。抗Vif抗体滴度可达1:204800,抗APOBEC3G抗体滴度可达1:102400。多克隆抗体可以检测到细胞内表达的相应抗原。结果表明,获得了高纯度的APOBEC3G及Vif融合蛋白,并制备了相应的高效价及特异性抗体,进一步证明了纯化后的重组蛋白具有良好的免疫原性和抗原性。

Abstract

To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 vif was amplified by PCR from plasmid of HIV-1 NL4.3 cDNA, and APOBEC3G gene was achieved by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E.coli cells, the accuracy of inserted gene and specificity of proteins were detected by two enzymes digestion technology, SDS-PAGE, and Western blotting. Rabbits were immuzied by Vif or APOBEC3G protein. Serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assay were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of anti-APOBEC3G antibodies was 1:102400. The antibidies could detect the antigen expressing in the cells. These fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved. These results ensure the immunogenicity and antigenicity of the purified recombinant proteins.

甜菜夜蛾核型多角体病毒IAP3在哺乳动物细胞中功能的研究

朱丽娜, 梅春蕾, 张忠信

2008, 23(3): 183 doi: 10.1007/s12250-008-2883-5

杆状病毒抑制细胞凋亡蛋白(inhibitor of apoptosis protein,IAP)在病毒的感染性和宿主范围上起重要作用。本文利用PCR方法,从甜菜夜蛾核型多角体病毒(SeMNPV)基因组中扩增出病毒的抑制细胞凋亡基因3(iap3),该基因克隆到pEGFP-C1质粒上,转化哺乳动物细胞HEK293,结果显示,甜菜夜蛾核多角体病毒IAP3的表达能明显抑制HEK293细胞由治疗肿瘤药物顺铂诱导的细胞凋亡。这首次证明了甜菜夜蛾核多角体病毒iap3在哺乳动物细胞中有抑制细胞凋亡的功能。

Abstract

The baculoviral inhibitors of apoptosis play a significant role in infectivity and viral host-range, which make them potential candidates for the engineering and improvement of baculovirus insecticidal. The iap3 gene of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), amplified by PCR, was 939 bp encoding IAP3. The PCR product was cloned into EcoR I /Bam H I of the plasmid pEGFP-C1. GFP was fused to the N-terminaus of IAP3 to study distribution in HEK293. It was observed that the plasmid expressing IAP3 significantly inhibited apoptosis induced by cisplatin in HEK293 cells. We conclude that the IAP3 of SeMNPV is functional in mammalian cells.

SYBR Green 荧光定量PCR检测猴免疫缺陷病毒血浆病毒载量

陆靖, 秦莉, 刘广杰, 赵思婷, 陈小平

2008, 23(3): 189 doi: 10.1007/s12250-008-2896-0

在猴免疫缺陷病毒(SIV)感染模型研究中,血浆病毒RNA载量常用作评价病毒感染进程及疾病状态的重要指标。本文以SYBR green 为染料,建立了一步法实时荧光定量PCR用于精确定量血浆病毒载量。该方法成功用于检测SIVmac251和SIVmac239感染的CEM×174细胞培养上清中的病毒RNA载量,检测下限为每管反应10 copies。在建立的SIVmac251感染恒河猴模型中,该方法用于检测猴血浆病毒RNA载量,其检测下限为每毫升血浆215 copies,并且病毒载量的变化曲线也与其它的报道相符。该方法具有较高的灵敏度和精度,并且较branched-chain DNA(b-DNA)和基于TaqMan探针的荧光定量PCR方法更为简单、易行。

Abstract

Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian Immunodeficiency Virus (SIV) infections. To accurately measure RNA viral loads, a one-step fluorescent quantitative assay was established based on SYBR green Real-Time reverse transcription-polymerase chain reaction technique (RT-PCR). The assay with a lower detection limit of 10 copies per reaction was successfully applied to quantification of SIVmac251 and SIVmac239 virus stocks produced on CEM×174 cells. Moreover, the performance of the SYBR green real-time PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that this technique can detect as less as 215 copies per milliliter of plasma and the dynamic pattern of viral load was similar with those based on other techniques from other reports. Our assay due to its convenience, sensitivity and accuracy could serve as a good alternative to branched-chain DNA (b-DNA) assay or real-time PCR assay based on TaqMan probes.

卡波氏肉瘤相关疱疹病毒orf65基因原核表达产物的免疫原性分析

付碧石, 李宝林, 王林定

2008, 23(3): 196 doi: 10.1007/s12250-008-2925-z

为了纯化卡波氏肉瘤相关疱疹病毒编码的小核衣壳蛋白并分析其免疫原性,我们在原核表达体系中表达了卡波氏肉瘤相关疱疹病毒编码的羧基端orf65基因产品。含重组质粒pQE-80L-orf65的大肠杆菌经由异丙基硫代-β-D-半乳糖苷诱导表达。融合蛋白通过亲和层析纯化。表达的蛋白和纯化的产品经由SDS-聚丙烯酰胺凝胶电泳鉴定。我们建立了一种ELISA方法来鉴定纯化的orf65的免疫原性。纯化的orf65蛋白用于免疫兔子,并获得抗血清。SDS-聚丙烯酰胺凝胶电泳显示纯化的orf65蛋白约为9 kDa,与预期一致。ELISA分析证明orf65具有免疫原性,orf65的多克隆抗体的免疫原性是对照血清中抗体的4倍以上。这些结果证明纯化的orf65蛋白具有很强的免疫原性,且可被用于筛查普通人群的KSHV感染.

Abstract

To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA

犬细小病毒的分离鉴定及其VP2蛋白在转基因烟草的表达

熊宁, 张勇, 王瑶, 杨宝玉, 陈士云

2008, 23(3): 203 doi: 10.1007/s12250-008-2928-9

从武汉一宠物医院患病犬粪便样品中分离了一株犬细小病毒(CPV)。经血凝试验、免疫胶体金试纸条检测、电镜观察以及PCR鉴定,该病毒属于CPV-2a血清型。在此基础上我们克隆了该病毒vp2基因5’端长度为1242bp的片段,并克隆到植物表达载体pBI121,采用农杆菌介导法转化烟草叶片,通过卡那霉素抗性筛选得到了转基因烟草。转化T0 代烟草进行Western blotting和T1代烟草的PCR检测证实vp2基因整合到烟草基因组并获得正确表达的VP2蛋白。

Abstract

A strain of canine parvovirus(CPV)was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5’ region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.

HSV-1核衣壳蛋白UL25与细胞内微管蛋白的相互作用及其生物化学分析

郭磊, 张莹, 车艳春, 吴文娟, 李卫中, 王丽春, 廖芸, 刘龙丁, 李琦涵*

2008, 23(3): 211 doi: 10.1007/s12250-008-2936-9

利用酵母双杂交筛选和半乳糖苷酶活性检测发现单纯疱疹病毒I型(HSV-1)核衣壳蛋白UL25与细胞骨架微管蛋白存在相互作用,对UL25蛋白细胞内荧光分析发现其定位于胞质中细胞骨架微管蛋白,进一步对其缺失突变体研究提示UL25蛋白在核内也发挥了功能。

Abstract

An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β–D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

HBV YVDD突变是广州地区重要的拉米夫定耐药类型

欧志英, 周荣, 何蕴韶

2008, 23(3): 218 doi: 10.1007/s12250-008-2963-6

目的:为了研究广州地区拉米夫定药物选择压力下HBV YMDD野生型和突变型的流行病学。
方法:利用流过式杂交原理和传统的反向斑斑点杂交技术,即流过式反向斑点杂交结合凯普DNA快速杂交仪检测HBV基因组中的YMDD野生型和突变型模序。
结果:检测了1021例拉米夫定用药治疗8个月以上的高度HBV拉米夫定耐药疑似病人血清。发现35.94%是单一感染,8.03% 为YMDD,7.93%为 YIDD,19.98%为YVDD; 64.06%是混合感染, YMDD和YIDD混合感染占1.96%,YMDD和YVDD混合感染占51.62%, YIDD和YVDD混合感染占1.96%, YMDD YIDD和YVDD混合感染占8.52%。包含YVDD的感染占82.08%。而治疗前的感染状态是:YMDD单一感染占36.93%,YIDD单一感染占6.07%,YVDD单一感染占17.04%。YMDD和YIDD混合感染占0.97%; YMDD和YVDD混合感染占33.99%; YIDD和YVDD 混合感染占0.98%; YMDD, YIDD和YVDD混合感染占4.02%。包含YVDD的感染类型只占56.03%。YMDD模序突变为YVDD模序的比率为34.32%,明显比YMDD模序突变为YIDD模序高(仅为10.97%,U=10.98, P<0.05)。
结论:拉米夫定药物选择下,广州地区HBV YVDD模序是进化的优势类型,是一种重要的耐药突变类型。

Abstract

The epidemiological effects of native and mutated YMDD motif in the HBV genome under the selective pressure of lamivudine were investigated. YMDD wild and mutation motif in HBV genome were detected by flow through reverse dot blots (FT-RDB) with KaiPuTM DNA HybriMax Rapid Hybridization Machine based on the principle of “Flow-through hybridization” and by the traditional Reverse Dot Blot assay. Sera from 1 021 suspected lamivudine-resistant chronic HBV carriers after more than 8 months of lamivudine therapy and the corresponding archived sera were collected and assayed. We found 35.94% were single type infections with 8.03% YMDD, 7.93% YIDD and 19.98% YVDD. It was also found that 64.06% were mixed infections including 1.96% YMDD and YIDD, 51.62% YMDD and YVDD, 1.96% YIDD and YVDD, 8.52% YMDD, YIDD and YVDD. The levels of infections containing YVDD motif reached 82.08%. The pretreatment infectious status were: YMDD single infection was 36.93%; YIDD single infection was 6.07%; YVDD single infection was 17.04%; YMDD and YIDD mixed infection was 0.97%; YMDD and YVDD mixed infection was 33.99%; YIDD and YVDD mixed infection was 0.98%; YMDD, YIDD and YVDD mixed infection was 4.02%. Infections containing YVDD motif were only 56.03%. The 34.32% mutation rate of YMDD motif to YVDD was significantly higher than the 10.97% of YMDD to YIDD (U=10.98, P<0.05), as estimated by Mann-Whitney U-test for non-parametric data. HBV containing YVDD motif might have an evolutionary ascendancy and become the dominant type under the selective pressure of lamivudine.

HPV16在乳腺癌中的表达及其致癌机制的研究

范春玲, 周景华, 胡成乙

2008, 23(3): 226 doi: 10.1007/s12250-008-2914-2

目的 为了研究人乳头状瘤病毒(HPV16)在乳腺癌中的表达及作用机理,检测了HPV16、诱导一氧化氮合酶(iNOS)、抑癌基因P53蛋白及人端粒酶逆转录酶hTERT在乳腺癌的表达及其间的相关性。 方法 应用免疫组化SP法共检测了52例乳腺癌和16例乳腺良性瘤HPV16、iNOS和P53及hTERT蛋白的表达。结果HPV16和P53及hTERT蛋白在乳腺癌组的阳性表达率均显著高于乳腺良性瘤组。统计分析表明,iNOS 、P53蛋白和hTERT蛋白表达阳性率与HPV感染率密切相关(P<0.05)。结论 HPV16感染参与了乳腺癌的发生,其致癌机制可能是通过诱导iNOS表达增加,产生NO,诱导p53基因突变,从而引起hTERT蛋白表达增加而导致乳腺癌的发生。

Abstract

To explore the role of Human papillomavirus (HPV) in mammary carcinogenesis, the expression of the HPV-16, iNOS , P53 and hTERT proteins in breast carcinomas and their relationships were investigated. 52 samples of breast cancer and 16 samples of benign breast tumors were assayed using the immunohistochemical SP method for detection of protein expression levels. The expression of HPV-16, iNOS, P53 and hTERT proteins in a mammary carcinoma was 44.2%, 57.7%, 63.5% and 59.6% respectively, which was significantly greater than the corresponding levels in the benign group. The expression of iNOS, P53 and hTERT was correlated with the presence of an HPV-16 infection in a mammary carcinoma(P<0.05). The connection between these events might also involve the iNOS, mutated type P53 and the hTERT protein.