Construction of Recombinant Adenovirus Co2expressing Human Wild2type p53, GM2CSF and B721 Genes

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 17 (3) : p53 GM2CSF B721,, (, ) 3, p53 GM2CSF B721 pbb2102 pbb2102 GT , BB ,, 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 , 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,,,, : , : Tel & Fax : (010) ,E2mail bmi ac cn ( DNA ),,1971 2,,, Received :August 22,2000 ;Accepted :October 17,2000,, 3 Corresponding author Tel & Fax : (010) E2mail bmi ac cn China Academic Journal Electronic Publishing House All rights reserved

2 , PAb1801 FITC B721 p53 GM2CSF B721, Pharmingen, GM2CSF ELISA Kit p53, Genzyme GM2CSF B , 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, DNA, T4 DNA (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 Π, 24 h LipofectAMIN Corp,Gene Therapy Unit) d (cytopathic (green fluorescence protein, GFP) effect,cpe),, (Ad2GFP), 3,2 500 rπmin 15 min,, [ ] [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 BB2102 pbb2102 (4 g) GT4050 (6 g) 293, (plaque assay) 5 7 d, PCR Ad2GFP BB2102, CsCl Hep22,, (pfuπml) DMEM,10 %,37,5 %CO Hep Sal Nde Hep22 6, Hind Xho Cla Hpa Not EcoR T4 Π ;24 h, DNA BioLab,DNA Klenow (multiplicity of infection,moi) Ad2GFP( 015 ml DMEM),MOI pfuπ, DMEM ;1 2 h, 2 ml ;48 h GFP China Academic Journal Electronic Publishing House All rights reserved

3 3 : p53 GM2CSF B Fig 1 Construction of BB2102 by homologous recombination in vivo BB2102, l Hanks, 20 l FITC p53 Hep22 Hep22 B721, 30 min, 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 ELISA BB2102 GM2CSF Hep22 Hep Π 60 mm,12 h BB2102 pbb2102 pbb2102,moi 50 pfuπ, DMEM 1 2 h, 2 d, 10 d GM2CSF ELISA GM2CSF, DNA, BB2102 B721 E2b DNA, Hep22 Hep Π 60 mm,12 h BB2102,MOI 50 pfuπ, DMEM 1 2 h, 48 h Ad2GFP BB %, Hanks 2 Hanks 2, 1 2 ml Hanks, B721 2, B721 p53πgm2csf pbb2102 pbb2102, GT ,10 15 d CPE,, CPE, PCR p53 GM2CSF B721, p53 GM2CSF B721, BB2102,, China Academic Journal Electronic Publishing House All rights reserved

4 332 17, 110 A 260 = , Ad2GFP BB2102 ( Πml) ;Ad2GFP BB2102 A 260 / A , A 260 ΠA , ; Ad2GFP BB2102 (pfuπml) 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 B GM2CSF production was determined from Hep22 cells at 24 hours intervals by ELISA China Academic Journal Electronic Publishing House All rights reserved

5 3 : p53 GM2CSF B ; 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 China Academic Journal Electronic Publishing House All rights reserved

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