(HR P ) CD 4 + ) L yt2 ( CD 8 + ) F ITC

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1 1999; 17 (6) Ch inese Journal of Parasito logy and Parasitic D iseases 325 FCC1gHN CSP : pcdna 3, (H el a ) (CSP), BALBgc : PfCSP H el a,, EL ISAW estern blotting T N K T, BALBgc : EL ISA ; W estern blotting 3813 kd a ; CD 4 + CD 8 +, N K : pcdna 32PfgH el a, N K H el a, (CSP ) [ 13 ] CSP, X,,, DNA, ; ( ) ;, (CM V ) pcdna 3, (H el a) PfCSP,, BALB gc 1 FCC1gHN 2 BABL gc 201 g, 6 8, 3 3, 3, 5, 5 2 (TM B ) Boeh ringer M annheim, A (ConA ) Sigm a, R PM I 1640 Gibco, 3 (CMB) (No ) (M T T ) Serva, (HR P ) IgG L 3T 4 ( CD 4 + ) L yt2 (CD 8 + ) F ITC IgG 4 PfCSP BALBgc H el a, YA C21 1PfCSP H el a pcdna 32CSP H el a 50 m l, R PM I , 5% CO 2 (M 5 ), H el a H el a, G418 (400 Λggm l), G418, G418 C1C3C5 C7 2H el a 4,, 32%,, g 20 m in, 100 Λl ddh 2O, SD S2PA GE,, 3 C3, SD S2 PA GE,, - 40

2 326,, 4 11, BALBgc, 40 Λg, 2 w k, 2 w k 20 Λg, d6,, 5(DAD ) 1% (0102 molgl ph 712 PBS ) 3 mm, 1, 6 6, 3 mm, 5 mm, 10 Λl (30 Λg), 12164, 37,, 6(EL ISA ), 200 Λl (5 Λg) 4 ph 714, 011% BSA 2PBS2Tw een20 3,, Λl,, 371 h, HR P2 IgG ( 9709, ), 200 Λl, 3730 m in,, TM B 200 Λl, 20 m in, 50 Λl 2 molgl H 2SO 4 OD 450 nm, OD 3 7(W estern blotting) SD S2 PA GE,, (N C) 1%BSA TBS, 37 2 h 014% 30 s,, 4 mm,, 1200, 371 h, TBS 3, 0105% TBS2T riton 1,, 1400 HR P IgG 371 h, TBS 3 ; DAB,, 8T, 75% 1 m in,, H ank s,, R PM I gm l, 014%, > 95% gm l 24, 1 m l, ConA (5 Λg 10 Λg) (5 Λg),, 37 5% CO 2 68 h, M T T 50 Λl, 37CO 2 4 h, 1 m l,,, OD 570 nm, OD 630 nm 9(N KC) [ 4 ], gm l (EC), 48 h YA C21, gm l, gm l, (TC), 014% > 95% 200 Λlg24, EC TC , R PM I 1640, 600 Λlg, 3, R PM I % CO 2 44 h, M T T (5 m ggm l) 50 Λlg, 4 h, 1 m l,, OD 570 nm, N KC (% ) = N KC : OD E+ OD T- OD E+ T 100% OD T 10, T ris2n H 4C l, 200 Λl (510 6 gm l), 100 Λl L 3T 4 (CD 4 + ) L yt2 (CD 8 + ), H ank s 3730 m in, H ank s 3,, F ITC IgG 100 Λl 110, m in, H ank s 3,,, 3 PfCSP HeLa pcdna 32PfCSP H el a, SD S kd a, C3, (2) 912% C3

3 1999; 17 (6) Ch inese Journal of Parasito logy and Parasitic D iseases SDS-PAGE 1HeLa 2C1 3C3 4C5 5C7 62SDS-PAGE (C3) 3W estern blotting M : 1, 23, 4HeLa : 14 F ig. 1SDS-PAGE analysis of the expressed products Lane 1The supernatant of normal HeLa cell l inelane 2The supernatant of C1 Lane 3The supernatant of C3Lane 4The supernatant of C5Lane 5The supernatant of C7Lane 6Protein molecular weight standard F ig. 2The result of SDS-PAGE scann ing (C3) F ig. 3Result of W estern blotting M Protein molecular weight standardag: 1, 2 Pur if ied expression protein3, 4The supernatant of normal HeLa cell l ine1-4immun ized mouse serum,,, PA GE m ggm l DAD EL ISA PfCSP w k, 116 EL ISA,,, W estern blotting 3813 kd a (2), H el a T M T T, Λg ConA OD ( nm ) , , t, (P > 0105), (P < 0101), OD , t, (P < 0101), NKC 5, N KC , t, (P < 0105),, (P > 0105) 4M TT 5 F ig. 4Transformation of lymphocytes measured by M TT assay F ig. 5Detection of natural killer cell activity, CD 4 + CD 8 +, t, (P < 0105), CD 4 + g CD 8 +, (P > 0105)

4 328 6CD 4 + T 7CD 8 + T F ig. 6Detection of mouse spleen CD 4 + T cell F ig. 7Detection of mouse spleen CD 8 + T cell,, CSP [ 5 ], FCC1gHN CSP g g, pcdna 3 CM V pcdna 32CSP, H el a, CSP,, BALBgc pcdna 3 (CM V ), SD S2PA GE, 3813 kd a, 3714 kd a, BALB gc,,,, ;, [ 6, 7 ] N KC, M HC M T T PfCSP N KC ; IFN N KC N KC CD 4 + CD 8 + T, CSP CD 4 + CD 8 + T, CD 4 + CD 8 + T [ 8, 9 ] CD 4 + T Kupffer g M HC, IFN 2Χ IL 22 CD 8 + T g M HC, IFN 2Χ [ 10, 11 ] PfCSP,,, CD 4 + CD 8 + T,,,,,, pcdna 32PfCSPgH el a,, CSP, DNA,,

5 329 1Yoshida N, N ussenzw eig RS, Potocujak P, et al. Hybridom a p ro2 duces p rotective antibodies directed against the sporozoite stage of m alaria parasite. Science 1980; Schodel F, W irtz R, Peterson D, et al. Imm unity to m alaria elicited by hybrid hepatitis B virus core particles carrying cirum 2 sporozoite p rotein ep itopeṡ J Exp M ed 1994; 180 (9) N ardin EH, N ussenzw eig RS. T cell responses to p re2erythrocytic stages of m alaria: role in p rotection and vaccine developm ent a2 gainst p re2erythrocytic stageṡ A nn Rdv Imm unol 1993; P lum b JA, M ilroy R, Kay SB. Effects of the PH dependence of 32( 4, 52dim ethythiazol222yl) 22, 52diphenyl2tetrazolium brom ide2 form azan absorp tion on chemosensitivity. Cancer Res 1989; Dam e JB, W illiam s JL, M ccutchan TF, et al. Structure of the gene encoding the imm unodom inant surface antigen on the sporo2 zoite of the hum an m alaria parasite P lasm od ium f alciparum. Science 1984; Stoute JA, SlaouiM, Heppner DG, et al. A p relim inary evalua2 tion of a recom binant circum sporozoite p rotein vaccine against P lasm od ium f alciparum m alaria. N Engl J M ed 1997; 336 (2) Rom ero P, M aryanski JL, Corradin G, et al. Cloned cytotoxic T cells recognize an ep itope in the circum sporozoite p rotein and p ro2 tect against m alaria. N ature 1989; Schofield L. T cell imm unity to m alaria sporozoiteṡ Exp Parasitol 1989; Goods M F. A m alaria vaccine strategy based on the induction of cellular imm unity. Imm unol Today 1992; Hoffm an SL, Isenbager D, Long GW, et al. Sporozoite vaccine induces genetically restricted T cell elim ination of m alaria from hepatocyteṡ Science 1989; 244 (4908) W eissw R,M ellouk S, houghten RA, et al. Cytotoxic T cells rec2 ognize a pep tide from the circum sporozoite p rotein on m alaria2in2 dected hepatocyteṡ J Exp M ed 1990; ( : ) A IM : STUD IES ON IMM UNE RESPONSES IN M ICE TO EXPRESSED PROD UCTS FROM CSP GENE OF PLASM OD IUM FALCIPARUM FCC1gHN ISOLATE 3 FAN G Zheng 33, YU X inbing, L IU Yanw en, LUO Shuhong Institute of P arasitology, S un Y at2s en U niversity of M ed ical S ciences, Guangzhou ABSTRACT To con struct the eukaryo tic exp ression system w ith pcdna 32PfCSPgH el a fo r CSP gene of P lasm od ium f alcip arum, and to observe the imm une responses in BALBgc m ice induced by the exp ressed p roteinṡ M ETHOD S: T he recom binant p lasm id pcdna 32PfCSP w as transform ed into m amm alian cell line of hu2 m an H el a cellṡ T he exp ressed p rotein w as isolated and analyzed by SD S2PA GE and used for imm unization of BALB gc m ice by subcu taneou s, in travenou s o r in traperitoneal adm in istration, respectively. EL ISA, W estern blotting, T lymphocyte p roliferation test, natural k iller cell activity and CD 4 + and CD 8 + T cells detection w ere used for observation of the level of humoral and cellular imm une responseṡ RESUL TS: Imm une sera strongly reacted w ith the exp ressed p rotein. T he titer of the antibodiesw as up to by EL ISA. W estern b lo tting analysis revealed a specific band at 3813 kd a. T he specific p ro liferative respon se w ith imm un ized BALB gc m ice sp leen cells w as rem arkablely h igher than that w ith control oneṡ T he ratio of CD 4 + and CD 8 + T cells and the N K cell activity w ere significantly h igher in the imm unization group than that in the control group ṡ CONCL USION: T he humoral and cell2m ediated imm une responses and elevated N KC activity to ex2 p ressed p roductsw ith the eukaryo tic exp ression system can specifically be detected in BALB gc m ice to generate, indicating that the exp ressed p rotein could enhance imm une activity in m ice. spon se Key words: P lasm od ium f alcip arum, circum sporozoite p rotein, H el a cell, exp ressed p roduct, imm une re2 3 Supported by the Ch inese M edical Board (CM B ) Foundation (N o ) 33 P resent address: D epartm ent of Parasitology, N antong M edical College, N antong