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.
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。
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.
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。
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.
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。
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.
2008, 23(3): 196 doi: 10.1007/s12250-008-2925-z
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
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。
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.
2008, 23(3): 211 doi: 10.1007/s12250-008-2936-9
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.
2008, 23(3): 218 doi: 10.1007/s12250-008-2963-6
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）。
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.
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。
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.
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