ISSN 100727626 CN 1123870ΠQ 2001 6 Chinese Journal of Biochemistry and Molecular Biology 17 (3) :329 334 p53 GM2CSF B721,, (, 100850) 3, p53 GM2CSF B721 pbb2102 pbb2102 GT4050 293, BB2102 293,, ELISA, BB2102 p53 GM2CSF B721 Hep22,BB2102 Hep22, 2 4 d,, 10 d, p53, B721, Q75,R73 Construction of Recombinant Adenovirus Co2expressing Human Wild2type p53, GM2CSF and B721 Genes QIU Zhao2hua,LAO Miao2fen,WU Zu2ze 3 ( Institute of Radiation Medicine,Academy of Military Medical Sciences, Beijing 100850, China) Abstract In order to develop a combined gene therapy paradigm for the treatment of cancer,a recombinant adenoviral shuttle plasmid pbb2102 containing human wild2type p53, GM2CSF and B721 genes was constructed by molecular cloning Recombinant adenovirus BB2102 was constructed by homologous recombination of pbb2 102 and adenoviral packaging plasmid GT4050 in 293 cells Adenoviruses with high titer and purity were ob2 tained by amplifying in 293 cells on a large scale and ultra2centrifugating in CsCl step gradient solutions The expressions of p53, GM2CSF and B721 genes in human laryngeal epithelial carcinoma cells Hep22 were deter2 mined respectively by immunohistochemical assay, ELISA and flow cytometric analysis The results showed that human wild2type p53, GM2CSF and B721 genes could be efficiently expressed in Hep22 cells mediated by BB2 102 The peak expression period was on the second to fourth day after infection and then tended to decrease However,on the tenth day a relatively low expression could still be detected These results provided a basis for further experimental and clinical studies of combined gene therapy for laryngeal and other cancers Key words adenovirus, tumor suppressor gene p53, granulocyte2macrophage colony2stimulating factor, co2 stimulatory factor B721,gene therapy,, DNA,,,, :2000208222, :2000210217 3 Tel & Fax : (010) 68158311,E2mail :wuct @nic bmi ac cn ( DNA ),,1971 2,,, Received :August 22,2000 ;Accepted :October 17,2000,, 3 Corresponding author Tel & Fax : (010) 68158311 E2mail :wuct @nic bmi ac cn 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
, PAb1801 FITC B721 p53 GM2CSF B721, Pharmingen, GM2CSF ELISA Kit p53, Genzyme GM2CSF B721 112, 11211 pbb2102 [2 ] p53 GM2CSF B721 GT4138 Sal, GT8020 Sal BB2102, 3 Nde,37 4 h, 1 l DNA Klenow 2 l 2 mmolπl dntp,37 20 min, 1 111 DNA, T4 DNA 15 11111 (1) GT8020 : B721 DH5, ; (2) pxc53 : B721, p53 B721 p53πgm2csf ; (3) GT4138 : p53, pbb2102 (granulocyte2macrophage colo2 ny stimulating factor,gm2csf) Hpa Nae pbb2102,, p53 cdna pbb2102 CsCl GM2CSF cdna 5 pbb2102 GT4050 DNA (internal ribosome entry site,ires) ; (4) GT4050 :5, BB2102 Fig 1 E1 E3 : 293 6, (Baxter Healthcare 415 10 5 Π, 24 h LipofectAMIN Corp,Gene Therapy Unit) 11112 10 20 d (cytopathic (green fluorescence protein, GFP) effect,cpe),, - 80 37 (Ad2GFP), 3,2 500 rπmin 15 min,, 330 17 [1 11113 293 ] [3 E1 ] 293, American Type Culture Collection (ATCC), DMEM,10 %, 260 nm (fetal bovine serum,fbs),37,5 %CO 2 ; 280 nm ( Πml) Taq DNA dntp Promega, PCR (Advantage TM PCR2Pure Kit) Clontech,DMEM (LipofectAMIN) Gibco BRL,, (SP Kit) DAB Zymed, p53 Advantage TM PCR2Pure Kit GT4138 GT8020 B721 Ase EcoR Hind SnaB Xho Not 11212 BB2102 pbb2102 (4 g) GT4050 (6 g) 293, (plaque assay) 5 7 d, PCR Ad2GFP BB2102, CsCl Hep22,, (pfuπml) DMEM,10 %,37,5 %CO 2 11213 Hep22 11114 Sal Nde Hep22 6, 5 10 5 Hind Xho Cla Hpa Not EcoR T4 Π ;24 h, DNA BioLab,DNA Klenow (multiplicity of infection,moi) Ad2GFP( 015 ml DMEM),MOI 20 40 60 80 100 pfuπ, DMEM ;1 2 h, 2 ml ;48 h GFP 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
3 : p53 GM2CSF B721 331 Fig 1 Construction of BB2102 by homologous recombination in vivo 11214 BB2102, 100 200 l Hanks, 20 l FITC p53 Hep22 Hep22 B721, 30 min, 5 10 4 Π 12,12 h Ad2GFP BB2102 ( 015 ml DMEM),MOI 50 pfuπ, DMEM 1 2 h, 2 ml 48 h P53, 211 BB2102 p53 PAb1801, DAB 11215 ELISA BB2102 GM2CSF Hep22 Hep22 5 10 5 Π 60 mm,12 h BB2102 pbb2102 pbb2102,moi 50 pfuπ, DMEM 1 2 h, 2 d, 10 d GM2CSF ELISA GM2CSF, DNA, 11216 BB2102 B721 E2b DNA, Hep22 Hep22 1 10 6 Π 60 mm,12 h BB2102,MOI 50 pfuπ, DMEM 1 2 h, 48 h Ad2GFP BB2102 293 0125 %, Hanks 2 Hanks 2, 1 2 ml Hanks, B721 2, B721 p53πgm2csf pbb2102 pbb2102, GT4050 293,10 15 d CPE,, CPE, PCR p53 GM2CSF B721, p53 GM2CSF B721, BB2102,, 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
332 17, 110 A 260 = 111 10 12, Ad2GFP BB2102 ( Πml) 812 10 12 212 10 12 ;Ad2GFP BB2102 A 260 / A 280 1133 1131, A 260 ΠA 280 113, ; Ad2GFP BB2102 (pfuπml) 111 10 11 214 10 10 212 Ad2GFP Hep22,, Fig 2 :,, MOI 50 pfuπ, 90 %, (MOI 100 pfuπ ),, infection at various MOI, 50 pfuπ, Fig 2 Transfer efficiency of Ad2GFP in Hep22 cells 48 hours after Fig 3 Immunocytochemical staining of the p53 protein in Hep22 cells 48 hours after infection with medium alone (A),Ad2GFP(B) or BB2 102 (C) 213 BB2102 p53 GM2CSF B721 BB2102 p53 Hep22, Fig 3 Ad2GFP ; BB2102,BB2102 p53 Hep22 BB2102 Hep22, ELISA GM2CSF Fig 4 : 2 4 d GM2CSF,, 10 d GM2 Fig 4 The expression of GM2CSF in Hep22 cells infected with BB2 CSF GM2CSF : ; BB2102 Hep22 BB2102 Hep22, B721, FITC B721 Fig 5 : Hep22 B721 102 GM2CSF production was determined from 1 10 5 Hep22 cells at 24 hours intervals by ELISA 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
3 : p53 GM2CSF B721 333 ; BB2102 (MOI 50 pfuπ ),2 d 90 % B721, T :BB2102 B721 Hep2 Fig 5 Immunofluorescent flow cytometry analysis for the expres2 sion of B721 in falsely infected (A) or BB21022infected (B) Hep22 cells FL12H(fluorescence 12height) :fluorescence intensity of anti2b7212 FITC;All :Total cells ;M1 (marker 1) :positive cells ; % Gated :per2 centage of positive cells ; Geo mean :geometric mean of fluorescence intensity ;Peak Ch :Fluorescence intensity of peak channel 3 T : MHC2 T (T cell receptor,tcr) ; T, B7 T CD28 IL22, [7,8 T ] GM2CSF ( antigen presenting cell, APC), GM2CSF,, GM2CSF, APC [9,10 ], ( IL22 B721 GM2CSF ),, [11 13 ], B721 GM2CSF, B721 GM2CSF,, [14 ] p53,, Ad2GFP BB2102 GFP : (1), (report gene), G1 ; (2) Hep22, p53 p53 p53 p53 B721 GM2CSF, P53 [4,5 ] p53,, P53, 2 4 d, [6 ], 2 d p53 p53, p53 B721, GM2CSF,, p53,, 2 4 d,, 10 d 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
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