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畜牧兽医学报 2010年第S1期41卷 70-75页

作者： LI Jiong LIU Yan-hong AN Fang-lan LIU Jun-lin LIU Xiang-tao SHANG You-jun YIN Hong State Key Laboratory of Veterinary Etiological Biology Key Laboratory of Animal Virology of Ministry of AgricultureLanzhou Veterinary Research InstituteChinese Academy of Agricultural SciencesLanzhou 730046China

We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into *** recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the *** pseudovirus was used to infect BHK-21 cells and resistant cells were screened with *** fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect *** recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed *** capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 *** mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.

关键词： retroviral vector FMDV capsid precursor protein gene green fluorescent protein gene BHK-21 cell

Membrane immunoglobulin expressed by retroviral vector gene transfer mimics partial function of the B-cell receptor in vivo

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Science China(Life Sciences) 2016年第9期59卷 49-58页

作者： Jing Lu Feng Chen Zhen Xu Lingling Zhang Peng Xu Depei Liu Chihchuan Liang State key Laboratory of Medical Molecular Biology Department of Biochemistry & Molecular Biology Institute of Basic Medical SciencesChinese Academy of Medical Sciences and Peking Union Medical College

Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell re- ceptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, result- ing in the phosphorylation of Syk and Erkl/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.

关键词： BCR membrane immunoglobuUn retroviral vector

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Effects of recombinant retroviral vector mediated human insulin like growth factor-1 gene transfection on skeletal muscle growth in rat

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Chinese Medical Journal 2006年第23期119卷 1991-1998页

作者： RONG Shu-Ling LU Yong-Xin LIAO Yu-Hua WANG Xiao-Lin GUO He-Ping CHANG Chao GAO Yan-Zhang MI Shao-Hua Wan Jian-Ping Department of Cardiology Union Hospital Tongji Medical CollegeHuazhong University of Science and Technology Wuhan 430022China Department of Paediatrics Heping Hospital Changzhi Medical College Changzhi 046000 China

Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hlGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochernistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghlGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth. Results Recombinant retroviral plasmid vector pLghlGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening, hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast （P〈 0.05）. The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast （P〈 0.05）. Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myobiast group （P〈 0.05）. Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.

关键词： insulin like growth factor-1 skeletal muscle growth retroviral vector

Knockdown of Human p53 Gene Expression in 293-T Cells by retroviral vector-mediated Short Hairpin RNA

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Acta Biochimica et Biophysica Sinica 2005年第11期37卷 779-783页

作者： De-Long HAO Chang-Mei LIU Wen-Ji DONG Huan GONG Xue-Song WU De-Pei LIU Chih-Chuan LIANG National Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100005,China NationalLaboratoryofMedicalMolecularBiology InstituteofBasicMedicalSciencesChineseAcademyofMedicalSciences

RNA interference(RNAi)is an evolutionarily conserved process of gene silencing in multipleorganisms,which has become a powerful tool for investigating gene function by reverse ***,many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAiin mammalian cells by transfection,but this is still limited in its application,especially when it is necessary togenerate long-term gene silencing in *** circumvent this problem,retrovirus- or lentivirus-deliveredRNAi has been ***,we described two retroviral systems for delivering short hairpin RNA(shRNA)transcribed from the H1 *** results showed that retroviral vector-mediated RNAi cansubstantially downregulate the expression of human p53 in 293-T ***,the retroviral vector-mediated RNAi in our transduction system can stably inactivate the p53 gene for a long *** toshRNAs transcribed from the U6 promoter,H1-driven shRNA also dramatically reduced the expression *** p53 downregulation efficiencies of H1-and U6-driven shRNAs were almost *** resultsindicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.

关键词： retroviral vector RNA interference p53

CONSTRUCTION AND IDENTIFICATION OF retroviral vector EXPRESSING HUMAN INTERLEUKIN-17 GENE

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Chinese Journal of Cancer Research 1997年第4期9卷 63-66页

作者：曹雪涛黄欣万涛赵忠良

Human interleukin 17(IL17) is a newly found cytokine secreted by activated T lymphocytes. Many of its characteristics remain unknown. To provide a way for understanding its role in immunology and hematology further, we constructed a recombinant retroviral vector pL17SN containing the human IL17 gene about 1.3kb with the coding region. The constructed retroviral vector packaged in CRIP cells could infect human primary fibroblasts, and could stimulate the primary fibroblasts secreting human GMCSF and IL6. The retroviral vector containing the human IL17 gene we constructed may present an efficient way to understand the biological functions of human IL17 and to investigate applications of IL17 in cancer gene therapy.

关键词： Interleukin17 retroviral vector Gene expression GMCSF Interleukin6.

Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection

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Chinese Medical Journal 2004年第9期117卷 1364-1369页

作者：李克强李文林彭淑牖石小玉唐洪林刘颖斌 Department of General Surgery Second Affiliated Hospital College of Medicine Zhejiang University Hangzhou 310009 China Jiangxi Key Laboratory of Medical Biotechnology Jiangxi Medical Science Institute Nanchang 330006 China Basic Medical Department of Jiangxi Medical college Nanchang 330006 China

Background This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene *** ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG 2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG 2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG 2-ANGPTL4 cells in vivo and in vitro, *** The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4×106 infective viral grains/ml, and the rate of HepG 2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG 2-ANGPTL4 cells than in HepG 2 cells. The expression of ANGPTL4 mRNA in HepG 2-ANGPTL4 cells was higher than in HepG 2-pMSCV cells (154% higher) or HepG 2 cells (161% higher). The proliferation rate of HepG 2-ANGPTL4 cells in vitro was obviously lower than those of HepG 2-pMSCV cells and HepG 2 cells (P<0.01). The mean volume and weight of tumors seeded from HepG 2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG 2 cells and HepG 2-pMSCV cells (P<0.01).Conclusion A stabl

关键词： angiopoietin-like 4 · HepG 2 cell · retroviral vector

Construction of RNAi recombinant retroviral vector of 14-3-3σand establishment of its stably transfected HaCat cell lines

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Construction of RNAi recombinant retroviral vector of 14-3-3...

Construction of retroviral vectors Containing Rat Fas Ligand Gene and FasL Expression Mediated by the vectors in Hepatocellular Carcinoma Cells

第七届中国核学会“三核”论坛中国放射医学教育五十年暨中国毒理学会放射毒理委员会第八次全国会议

作者： Zhou Meijuan Ding Zhenhua Department of of Radiation Medicine Public Hygiene and Tropical Medicine SchoolSouthern Medical University

Objective To construct the RNAi retroviral vector of 14-3-3σand establish the stable transfect- ed HaCat cell *** Hairpin siRNA of 14-3-3σwere chemically synthesized and inserted into pSuperretro -neo-EGFP ***-retro-neo-EGFP-si14-3-3σwere transformed into competent STBL3 cells to *** the positive clones were confirmed by sequencing and transfected into the packaging 293FT cells to generate,amplificate and depurate *** ceils were infected with the recombinant retroviral vector and the expression of 14-3-3σwas detected by Western blot and real time *** The recombinant retroviral plasmid PSuper-retro-neo-EGFP-si14-3-3σwas successfully constructed,and green fluorescence of the stable transfected HaCat cell lines could be observed under inverted fluorescence *** results of western blot and real time PCR showed that the sequence of RNAi-14-3-3σcould effectively down-regulate the level of *** We have successfully constructed the RNAi retroviral vector targeting 14-3-3σand have established stably transfected HaCat cell lines.

关键词： 14-3-3σ retroviral vector stably transfected HaCat cell lines

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The Journal of Biomedical Research 2001年第1期15卷 13-21页

作者：孙倍成王学浩钱建民张峰许志祥李相成 Department of Hepatobiliary Surgery the First Affiliated Hospital Nanjing Medical University Institute of Biotechnique Suzhou Medical College

Objective In order to study the biological function of Fas and Fas ligand system, discuss the feasibility of treating tumor with transfecting FasL gene. Methods the rat Fas ligand complementary DNA was subcloned to retroviral vector pLXSN, acquired pLXSN/FasL + recombinant with direct inserting and single copy,then packaged with PA317 amphotropic packaging cells,anti G418 clones were acquired,and it was named PA317/ pLXSN FasL + *** The titer of virus was 4.7×10 7 CFU/ml,there was FasL gene integration in PA317/pLXSN FasL + cells detected by polymerase chain reaction. When we used the supernatant of the PA317/pLXSN FasL + cells to infect hepatocellular carcinoma cells HepG 2,SMMC 7721,CBRH 7919 and RH 35 ,the FasL expression was found at all the surface of the four cell lines through FCM,and the apoptosis in HepG 2 and CBRH 7919 cells which had high levels Fas expression was found *** the results show that it is an effective way to introduce FasL gene to retroviral vectors, which can be used to induce apoptosis in the cells with high levels Fas expression.

关键词： FasL gene retroviral vector apoptosis hepatocellular carcinoma

Overexpression of heme oxygenasel protects smooth muscle cells against oxidative injury and inhibits cell proliferation

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Cell Research 2002年第2期12卷 123-132页

作者： MIN ZHANG, BAO HuI ZHANG, LI CHEN, WEI AN1 Institute of Sports Medicine, The Third Hospital, Peking University, Beijing 100083, China 2Department of Cell Biology, Capital University of Medical Sciences, Beijing 100054, China 1Institute of Sports Medicine The Third Hospital Peking University Beijing 100083 China 1Institute of Sports Medicine The Third Hospital Peking University Beijing 100083 China 2Department of Cell Biology Capital University of Medical Sciences Beijing 100054 China 2Department of Cell Biology Capital University of Medical Sciences Beijing 100054 China

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of H

关键词： Heme oxygenase retroviral vector free radical cell proliferation vascular smooth muscle cells.

Expression of YB-1 enhances production of murine leukemia virus vectors by stabilizing genomic viral RNA

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Protein & Cell 2012年第12期3卷 943-949页

作者： Wei Li Xinlu Wang Guangxia Gao Key Laboratory of Infection and Immunity Institute of BiophysicsChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China

Murine leukemia virus(MLV)-based retroviral vectors is widely used for gene transfer and basic research,and production of high-titer retroviral vectors is very *** we report that expression of the Y-box binding protein 1(YB-1)enhanced the production of infectious MLV ***-1 specifically increased the stability of viral genomic RNA in virus-producing cells,and thus increasing viral RNA levels in both producer cells and virion *** viral element responsive to YB-1 was mapped to the repeat sequence(R region)in MLV genomic *** results identified YB-1 as a MLV mRNA stabilizer,which can be used for improving production of MLV vectors.

关键词： Y-box binding protein 1 retroviral vector RNA stability