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1.
Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells. Methods: Seven truncated CORE-GFP (green fluorescent protein) fusion protein expression plasmids were constructed,which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; BNT: 1-172 aa and C191:1-172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry. Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1-172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT＞BNT＞C191). To the different fragment COREs of BT,N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1-172 aa＞1-126 aa＞1-58aa＞ 127-172 aa＞59-126 aa). Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of different HCV quasi-species might have some difference in their pathogenesis.  相似文献   

2.
增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),也称周期蛋白或DNA聚合酶的辅助蛋白,是真核细胞合成所必需的核蛋白,在DNA复制中起重要作用。前期实验发现日本七鳃鳗肝脏cDNA文库的表达序列标签(Expressed Sequence Tag,EST)中存在与高等脊椎动物pcna基因同源的序列。提取日本七鳃鳗(Lampetra japonica)肝脏组织RNA,通过RT-PCR方法扩增七鳃鳗pcna基因,对其进行生物信息学分析,并将Lj-pcna基因成功构建到pGFP-N2真核表达载体上,重组质粒PGFP-N2-Lj-pcna转染人Hela细胞,荧光显微镜下观察有荧光蛋白的表达。日本七鳃鳗pcna基因的真核表达载体成功构建和转染,为探讨七鳃鳗pcna基因功能研究及其它七鳃鳗相关研究提供条件。  相似文献   

3.
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4.
绿色荧光蛋白及其在分子生物学上的应用   总被引：3，自引：0，他引：3  
许多海洋无脊椎动物体内都含有绿色荧光蛋白,这种蛋白质结构很特殊,在受到蓝光或紫外线刺激时可以发射绿色荧光,并不需要任何协同因子、底物,适合用作普遍的报告标记,尤其适合于活体细胞或组织。而且它发出的荧光稳定,检测简单,结果真实可靠。并且GFP对光漂白、氧化剂、还原荆以及其他许多化学试剂具有极强的稳定性．易于构建载体。它独特的性质引起了生物学界的广泛关注。本文简要地概述了绿色荧光蛋白及其在分子生物学上的应用,包括蛋白融合,细胞筛选,定位标记,基因表达调控,计算细胞生长速度等。  相似文献   

5.
在人体内,CCR5与许多免疫疾病有关,CCR5有望成为众多药物的作用靶点。将ccr5基因与真核表达载体pBBS242构建成重组质粒pBBS242-ccr5,转染CHO细胞,并经潮霉素B筛选。流式细胞仪检测结果表明CCR5在CHO细胞得到了稳定表达。  相似文献   

6.
Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.  相似文献   

7.
目的:我们的目标是从源于胚胎体(EBs)的小鼠胚胎干细胞(mESCs)中分离运动神经元样细胞前体(MNLCPs),以用于发展针对运动神经元疾病的药物筛选试验和移植疗法.天然Shh蛋白(或Shh通路激动剂)和维甲酸诱导的胚胎体中,MNLCPs和未分化细胞的含量是不确定的.如果不把未分化的细胞从细胞培养中充分去除,其可能会干涉药物筛选试验或在移植后增生.我们开发了一种以密度梯度离心法为基础的富集MNLCPs的方法.方法:我们用Wichterle等人2008年改进的方法,将mESCs( HBG3:eGFP:HB9)扩大和分化.通过化学和酶学的无研磨处理,含有绿色荧光蛋白的MNLCPs和未分化细胞的胚胎体被小心轻轻地离解成单细胞.利用OptiprepTM8％～20％逐步梯度离心技术将MNLCPs回收.拥有绿色荧光蛋白的MNLCPs的含量由流式细胞仪检测.结果:我们的结果表明,在胚胎体形成前,mESCs在明胶包被的培养板上生长,其分化为MNLCPs的能力减少.比较mESCs在明胶,明胶与PMEFs,及PMEFs包被的培养板上的生长发现,mESCs在PMEFs包被的培养板上产生含绿色荧光蛋白的MNLCPs的得率为(54.1±11.0)％(x-±s;n=12),在明胶包被的培养板上的得率为(2.8±1.1)％(x-±s;n=9).用密度梯度离心法获得的含绿色荧光蛋白的MNLCPs的平均含量为(87.7±5.5)％(x-±s;n=3).结论:我们的数据表明,不使用细胞分选器,无研磨解离和密度梯度离心法也能用于富集具有高存活率的MNLCPs.有必要对MNLCPs在体外、体内和表型上进行进一步的生理学意义(如神经轴突的生长及形成神经肌肉接头的能力)上的鉴定.  相似文献   

8.
To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken  相似文献   

9.
Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments. Project (No. 001103058) supported by the Key Program of Science and Technology Bureau of Zhejiang Province, China  相似文献   

10.
To construct and identify further a recombinant of Adeno-associated virus and interferon-gamma for gene therapy, the full-length IFN-γcDNA containing signal peptide was amplified by PCR, and then cloned into the pUC18. After screening, the fragment from the positive clone was then subcloned into pwp19. After the correct recombinant was identified by digestion with SacI and BamHI, it was transfected into lymphocyte cell line H9 mediated by calcium phosphate, and the expression of IFN-γ was detected by RT-PCR and ELISA. The result showed that the IFN-γ were expressed in the H9 cells transfected with pwp/IFN-γ. The so constructed recombinant plasmid pwp19/IFN-γ containing the full-length IFN-γ gene was expressed in mammalian cells. Project (39570653) supported by NFSC.  相似文献   

11.
Objective： To investigate whether vascular endothelial growth factor （VEGF） gene plasmid carried by polytetrafluoroethylene （PTFE） vascular graft materials could transfect endothelial cells （ECs） and promote their growth. Methods： PTFE vascular graft materials carried with pCDI-hVEGF121, pCDI or pEGFP were incubated in Tris-buffer solution and the values of optical density of 260 nm at different time were plotted, then the DNA controlled release curve was made. ECs derived from human umbilical vein were seeded on the pCDI-hVEGF121/pCDI/pEGFP-PTFE materials or tissue culture plates, ECs numbers were counted and VEGF protein concentrations at different time were measured by enzyme-linked immunoadsorbent assay method. Green fluorescent protein （GFP） expression in ECs on pEGFP-PTFE materials was examined with fluorescence mi- croscopy. Results： The controlled release curve showed that the gene released from PTFE materials was rapid within 8 h, then slowed down and that the gene released continuously even after 72 h. At 24, 72 and 120 h, ECs number and proliferation rate of pCDI-hVEGFI21-PTFE materials were higher than those ofpCDI or pEGFP-PTFE materials （P〈0.05）. VEGF protein concentration of pCDI-hVEGF121-PTFE materials was higher than that of pC DI or pEGFP-PTFE materials at 6, 24, 72 and 120 h （P〈0.01）. GFP expression in ECs on the pEGFP-PTFE materials could be detected by fluorescence microscopy. Conclusion： PTFE graft can be used as a carrier of VEGF gene plasmid, VEGF gene carried by PTFE can transfect ECs and promote ECs growth.  相似文献   

12.
Objective: To construct a eukaryotic expression plasmid pcDNA3.1 (-)-Humanin. Methods: The recombinant plasmid pGEMEX-1-Humanin was digested with restriction endonucleases BamH I and Hind III and the Humanin gene fragments, about 100 bp length, were obtained. Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1 (-) and the recombinant plasmids pcDNA3, l(-)-Humanin were identified by sequencing. Results: Recombinant plasmid DNA successfully produced a band which had the same size as that of the Humanin positive control. The sequence of recombinant plasmids accorded with the Humnain gene sequence. Conclusions: A eukaryotic expression plasmid of Humanin was successfully constructed.  相似文献   

13.
Eukaryotic initiation factor subunit c(eIF3c) has been identified as an oncogene that is over-expressed in tumor cells and,therefore,is a potential therapeutic target for gene-based cancer treatment.This study was focused on investigating the effect of small interfering RNA(siRNA)-mediated eIF3c gene knockdown on colon cancer cell survival.The eIF3c gene was observed to be highly expressed in colon cancer cell models.The expression levels of the gene in eIF3c siRNA infected and control siRNA infected cells were compared via real-time polymerase chain reaction(PCR) and western blotting analysis.Cell proliferation levels were analyzed employing 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide(MTT) and colony formation assays.Furthermore,the effects of eIF3c gene knockdown on the cell cycle and apoptosis were analyzed using flow cytometry.The results showed that suppression of eIF3c expression significantly(P<0.001) reduced cell proliferation and colony formation of RKO colon cancer cells.The cell cycle was arrested by decreasing the number of cells entering S phase.Further,apoptosis was induced as a result of eIF3c knockdown.Collectively,eIF3c deletion effectively reduced the survival of colon cancer cells and could be used as a therapeutic tool for colon cancer therapy.  相似文献   

14.
INTRODUCTION Chronic infection by hepatitis B virus (HBV) isassociated with a high risk of liver cirrhosis and pri-mary hepatocellular carcinoma (HCC). It remains animportant global health problem with over 350 mil-lion chronic HBV carriers worldwide. Among thesechronic carriers, about one million people die ofHBV-associated liver failure or HCC annually (Kaoand Chen, 2002). Although chronically infected pa-tients have been treated with interferons and nucleo-side analogs, th…  相似文献   

15.
Objective: To explore the effects of cytomegalovirus (CMV) infection on rejection-related gene expression in the endothelial cells of renal transplantation recipients. Methods: Endothelial cells (ECs) were cultured and stimulated by a variety of factors: A, normal control group; B, inactivated human cytomegalovirus (HCMV) infection group; C, HCMV infection group; D, HCMV supematant infection group; and E, ganciclovir HCMV group. Expression of intercellular adhesion molecule-1 (ICAM-1) and major histocompability complex (MHC) class Ⅰ and class Ⅱ antigens was detected by flow cytometry (FCM) and immuno-histochemistry. Results: We found characteristic CMV-infected ECs in this study. There were no significant differences among groups A, B and D (P>0.05). Although the expression levels of ICAM-1 were not significantly different between groups C and E (P>0.05), the ICAM-1 expression in these two groups was significantly higher than that in group A (P<0.05). ICAM-1 expression was detected in groups C and E, while there was no expression in groups A, B and D. Furthermore, there was no significant difference of ICAM-1 mRNA expression between groups C and E (P>0.05). Human leucocyte antigen (HLA)-ABC expression was detected in all the groups, while HLA-DR expression was only detected in groups C and E. There were no significant dif-ferences of HLA-ABC and HLA-DR expression among groups A, B and D (P>0.05). However, the HLA-ABC and HLA-DR expression levels in groups C and D were higher than those of the remaining groups previously reported (P<0.05). Meanwhile, the HLA-ABC and HLA-DR expression levels in group E were lower than those of group C (P<0.05). Conclusion: CMV could up-regulate the expression levels of ICAM-1 and MHC antigens, which was closely related to allograft rejection.  相似文献   

16.
Objective: To study the relationship between plasma adiponectin concentration and the functional activities of circulating endothelial progenitor cells (EPCs) in patients with coronary artery disease (CAD). Methods: Circulating EPCs were enumerated as AC133+/KDR+ cells via flow cytometry and identified by co-staining with Dii-acLDL and fluorescein isothiocy-anate (FITC)-conjugated lectin under a fluorescent microscope. The migratory capacity of EPCs was measured by modified Boyden chamber assay. Adhesion capacity was performed to count adherent cells after replating EPCs on six-well culture dishes coated with fibronectin. Results: The number of circulating EPCs (AC133+/KDR+ cells) decreased significantly in CAD patients, compared with control subjects [(74.2±12.3) vs (83.5±12.9) cells/ml blood, P<0.0\]. In addition, the number of EPCs also decreased in CAD patients after ex vivo cultivation [(54.4±8.6) vs (71.9±11.6) EPCs/field, P<0.01]. Both circulating EPCs and differentiated EPCs were positively correlated with plasma adiponectin concentration. The functional activities of EPCs from CAD patients, such as migratory and adherent capacities, were also impaired, compared with control subjects, and positively correlated with plasma adiponectin concentration. Conclusion: The study demonstrates that the impairment of the number and functional activities of EPCs in CAD patients is correlated with their lower plasma adiponectin concentrations.  相似文献   

17.
目的:探测细胞氧化低密度脂蛋白(LDL)过程中人α-防御素-1(HNP-1)基因的表达水平并构建人HNP-1的克隆载体。方法:提取人单核细胞系(THP-1)的总RNA,逆转录聚合酶链反应(RT-PCR)法获得cDNA,采用pBS-T克隆HNP-1。结果:用RT-PCR在THP-1细胞总RNA中扩增出一条285 bp的DNA片段,与HNP-1 cD-NA片段大小一致。重组质粒经PCR和测序鉴定获HNP-1基因克隆。另外,LDL可诱导THP-1细胞HNP-1的mR-NA表达增加。结论:HNP-1可能参与LDL的细胞氧化过程,成功构建HNP-1克隆载体将为进一步亚克隆到原核及真核的表达载体提供了基础。  相似文献   

18.
绿僵菌是一类广泛应用于生物防治的昆虫病原真菌.研究表明小RNA能够调控基因的表达,其中Argonaute基因在整个小RNA通路中发挥重要的作用.本研究通过RT-PCR的方法从绿僵菌中获得了Argonaute基因的部分功能片段,构建了其重组原核表达载体,将重组载体转化至大肠杆菌进行诱导表达;采用镍柱亲和纯化重组的目的蛋白并通过Western blot技术鉴定.结果发现：通过RT-PCR的方法获得长度约为950bp的基因片段;重组原核表达载体经诱导表达后,SDS-PAGE检测发现分子量约为34kDa的目的蛋白条带;诱导5h后蛋白的表达量最高,采用镍柱亲和层析纯化重组蛋白,经Western blot技术检测,重组蛋白可与His-tag抗体发生特异性反应.该纯化重组蛋白的获得为将来绿僵菌Argonaute蛋白抗体的制备,并进一步通过该抗体获得绿僵菌体内的小RNA及其靶基因提供了基础.  相似文献   

19.
Objective: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4~+ CD25~+ regulatory T (Treg) cells, in order to inform further studies in Treg cell function. Methods: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4~+ CD25~+ Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4~+ CD25~+ Treg cells, trypan blue staining to detect cell viability, and propidium iodide (PI) staining to assess the cell viability. We detected the immune inhibition of CD4~+ CD25~+ Treg cells in the in vitro proliferation experiments. Results: The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained than with flow cytometry sorting. The CD4~+ CD25~+ Treg cells, however, obtained with both methods have similar immunosuppressive capacities. Conclusion: The result suggests that both methods can be used in isolating CD4~+ CD25~+ Treg cells, and one can select the best method according to specific needs and availability of the methodologies.  相似文献   

20.
Lyu  Sunjian  Yuan  Xuemei  Liu  Li  Zhang  Haiqi  Yu  Zhe  Hang  Xiaoying  Shi  Weida  Wu  Yinglei 《Journal of Zhejiang University. Science. B》2021,22(4):295-304
Trionyx sinensis Hemorrhagic Syndrome Virus(TSHSV) is an arterivirus newly discovered in Chinese softshell turtles. Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles. In this study, a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system, and its polyclonal antibody was generated. The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay(dot-ELISA). The distribution of TSHSV in different organs of T. sinensis was examined by immunohistochemistry(IHC) and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction(qRT-PCR). The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed, and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles. The IHC assay indicated that the virus was highly localized in various cells, including intestinal lymphocytes,enterocytes, kidney epithelial cells, spleen cells, lung macrophages, and cardiomyocytes. The qRT-PCR analysis revealed that TSHSV was detected in all organs tested, including the lungs, liver, kidneys, spleen, and heart. The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group. The interferonstimulated genes(ISGs), myxovirus resistance protein 2(MX2) and radical S-adenosyl methionine domain containing 2(RSAD2) were highly upregulated in all groups of infected turtles. Antibody-dependent enhancement(ADE) seemed to occur after stimulation by the polyclonal antibody, because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group. Overall, these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.  相似文献   

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