|
|
|
题名
|
作者
|
年代
|
出处
|
被引量
|
| 1 | A rapid and efficient method to express target genes in mammalian cells by baculovirus显示文摘AIM: To investigate the modification of baculovirus vector and the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of Spodoptera frugiperta (Sf9) cells infected by recombinant baculoviruses. METHODS: Two recombinant baculoviruses (BacV-CMV-EGFPA, BacV-CMV-EGFPB) containing CMV-EGFP expression cassette were constructed. HepG2 cells were directly incubated with the culture supernatant of Sf9 cells infected by recombinant baculoviruses, and reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM). The optimal transduction conditions were investigated by FCM assay in HepG2 cells. Gene-transfer and expression efficiencies in HepG2 or CV1 cells by baculovirus vectors were compared with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Twenty different mammalian cell lines were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the culture supernatant of infected Sf9 cells. RESULTS: CMV promoter could directly express reporter genes in Sf9 cells with a relatively low efficiency. Target cells incubated with the 1:1 diluted culture supernatant (moi=50) for 12 h at 37 ℃ could achieve the highest transduction and expression efficiencies with least impairment to cell viability. Under similar conditions the baculovirus vector could achieve the highest gene-transfer and expression efficiency than lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Most mammalian cell lines could be transduced with recombinant baculovirus. In primate adherent culture cells the recombinant baculovirus could arrive the highest infection and expression efficiencies, but it was not very satisfactory in the cell lines from mice and suspended culture cells. CONCLUSION: Mammalian cells incubated with the culture supernatant of infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign genes in mammalian cells, but it might be more suitable for primate adherent culture cells. | TongCheng Chen-YuXu Ying-BinWang MinChen TingWu JunZhang Ning-ShaoXia | 2004 | World Journal of Gastroenterology2004，10，11： | 7 |
| 2 | Prokaryotical expression of structural and non-structural proteins of hepatitis G virus显示文摘AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or)pGEX. and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.``RESULTS One clone with HGV fragment from core to El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification.``CONCLUSION Core to El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis. | Ning-Shao Xia~1 Hai-Jie Yang~1 Jun Zhang~1 Chang-Qing Lin~1 Ying-Bin Wang~1 Juan Wang~1 Mei-Yun Zhan~2 MH Ng~3 1 Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering,Xiamen University,Xiamen 361005,Fujian Province,China2 Institute of Virology,Chinese Academy of Preventive Medicine Beijing 100052,China3 Department of Microbiology,Hoog Kong University,Hongkong,China | 2001 | World Journal of Gastroenterology2001，7，5： | 4 |
| 3 | HGV RNA转录体感染恒河猴的血清学及组织学改变显示文摘目的:观察接种HGV实验猴的血清学和组织病理改变,以探讨HGV对恒河猴的致病性。 方法:将庚型肝炎病毒(HGV)全长RNA转录体肝内注射感染恒河猴,检测血清HGV RNA、ALT变化和抗HGV,定期取实验猴肝组织,观察肝组织病理改变,免疫组化对HGV E2蛋白的表达进行定位分析,尸检取猴的各种脏器进行常规病理检查,并分析HGV E2蛋白在心、肝、胰、脾和肾脏的表达,PCP检测心、肝、胰、脾和肾脏中HGV正负链RNA的存在,透射电镜分析肝组织超微结构的变化。 结果:感染后1wk HGV RNA即阳转,最长持续达27wk,实验恒河猴血清ALT间歇性升高,最高达2106nkat·L^(-1),肝组织学活检呈现轻度炎性病变,尸检结果表明实验猴死于间质性肺炎,除肝组织炎症改变外,其他组织正常,免疫组化显示HGVE2蛋白主要在肝细胞质内表达,在心脏和脾脏亦有不同程度的表达,HGV基因组RNA和负链RNA的检测表明HGV在肝脏以外的其他脏器如心和脾脏中亦有复制,电镜结果表明肝细胞超微结构改变较大,肝窦内发现直径约25nm~30nm的病毒样颗粒呈晶格状排列。 结论:HGV RNA转录体有感染性并能形成病毒血症,恒河猴对HGV感染敏感,可作为研究HGV复制及致病性的动物模型。任浩,朱分禄,朱诗应,王路,戚中田,HGV RNA转录体感染恒河猴的血清学及组织学改变,世界华人消化杂志2002; | 任浩 朱分禄 朱诗应 王路 戚中田 | 2002 | 世界华人消化杂志2002，10，1： | 3 |
您所在的机构暂未开通此部分的功能，请联系管理员