1999; 17 (6) 325329 Ch inese Journal of Parasito logy and Parasitic D iseases 325 FCC1gHN CSP 3 33 510089 : pcdna 3, (H el a ) (CSP), BALBgc : PfCSP H el a,, EL ISAW estern blotting T N K T, BALBgc : EL ISA 16 400; 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. 932594) 33 226001 (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 1640 37, 5% CO 2 (M 5 ), H el a H el a, G418 (400 Λggm l), G418, G418 C1C3C5 C7 2H el a 4,, 32%,, 3 400 g 20 m in, 100 Λl ddh 2O, SD S2PA GE,, 3 C3, SD S2 PA GE,, - 40
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,, 150112 800 200 Λl,, 371 h, HR P2 IgG ( 9709, 11 000), 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 1640 5 10 6 gm l, 014%, > 95% 210 6 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 ], 510 6 gm l (EC), 48 h YA C21, 110 5 gm l, 01510 5 gm l, (TC), 014% > 95% 200 Λlg24, EC TC 50 1 25 1, R PM I 1640, 600 Λlg, 3, R PM I 1640 3750% 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, 37 30 m in, H ank s 3,,, 3 PfCSP HeLa pcdna 32PfCSP H el a, SD S 1 3813 kd a, C3, (2) 912% C3
1999; 17 (6) 325329 Ch inese Journal of Parasito logy and Parasitic D iseases 327 1 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 1 014 m ggm l DAD EL ISA PfCSP 47 12 w k, 116 EL ISA,,, 16 400 W estern blotting 3813 kd a (2), H el a T M T T, 4 5 10 Λg ConA OD (570630 nm ) 01370105 01400105, 0134 0103 01360104, t, (P > 0105), (P < 0101), OD 01340104 01120103, t, (P < 0101), NKC 5, N KC 251 501, 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, 6 75 7 CD 4 + CD 8 +, t, (P < 0105), CD 4 + g CD 8 +, (P > 0105)
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,,
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