HBV ( ) Vol. 47 No mab pres1 ( 21247) ., pres1. ,pres1. Dane 1011vge (viral genome equiva2 lent s). HBsAg,

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1 47 2 () Vol. 47 No Journal of Xiamen University (Nat ural Science) Mar HBV pres1,,,, 3 (,, ) : ( HBV) (Dane ) HBV S1 (pres1) GST2preS1 Balb/ c,7 (mab), EL ISA PCR. mab Ig G1 Ig G2a,. mab EL ISA, 7 pres1 mab HBV, mab 4D11 HBV. EL ISA pres1 (21247) B,mAb 4D11 1 G5 7B6 7 H11 B ; mab 3 H5 6F1 2A6. HBV pres1 HBV pres1,hbv pres1. : ; HBV ;pres1 ;B :Q :A : (2008) HBV, [ 1 ],. pres1 S2 HBV,.,preS1., pres1., HBV, [2 ]., 3 :1) pres1, A 6 [3-4 ] ;2) pres2, ( P HSAR) [5 ] ; 3) HBsAg, H (apolipoprotein H) V (annexin V) [6 ]., pres1 (21247) HBV ;pres1 (21247) HBV,preS1 HBV [ 7 ]. pres1 GST2preS1,,mAb, : : 863 (2006AA020905), (2004 YZ01) 3 edu. cn mab pres1 ( 21247) HBV. mab pres Dane, Dane 60 %, Dane 1011vge (viral genome equiva2 lent s). GST2preS1 ; PreS1 (212 47). Balb/ c ; Sp2/ 0. PEG1500 DM2 SO Sigma ;RPMI1640 Gibco ;Hyclone ; HRP2( GAM) Ig G ;A P GAM ( H + L ) Protos ; HRP2GAM IgM Ig G1 Ig G2a Ig G2b Ig G3 Serotec ; GAM 2UNLB GAM 2UNLB SBA ; TE2 CAN HBV pres1 mab :Dane Balb/ c,2 GST2preS1 5 g/

2 2 :HBV pres ,GST2preS1 1,5g/. [8-9 ],. ( EL ISA) pres1 (21247). [10 ]. D EA E mab EL ISA mabs. mab Ig HRP2GAM IgM Ig G1 Ig G2a Ig G2b Ig G3 ; mab HRP2GAM 2UNLB HRP2GAM 2UNLB., EL ISA mab PCR Eppendoff, Dane. Dane,1 %SDS 200 g/ ml K,56 2 h. Dane DNA,PCR. PCR :10 buffer 2L, T aq 0. 2L,dN TP 0. 2L,BvCF 0. 2 L,BvCR 0. 2L,25 mmol/ L MgCl2 1. 2L,dd H2 O 11L, 5L. :94 4 min, 94 1 min ;53 40 s ;72 30 s,30, min. HBc, : BvCF :5 2GGA TCCCA TA T GGACA TTGACCCA23 ( HBc :nt1215) BvCR :5 2GAA TTCTTAACA TTGA GA TTCCCG23 ( HBc :nt ) EL ISA mab HBV 10 9 vge/ Dane (,HBV ),37 2 h 4. PBST,37 2 h. (, ) 0. 1 ml, min,pbst ml min,pbst 5,. 1 pres1 mab Tab. 1 Isotype of the anti2pres1 mabs HBV mab [ 11] mab 4D11 mab 6F1 [12 ],10 9 vge/ Dane EL ISA, mab mab 4D11 mab 6F1. 50L pres1 mab 50L,H EV E1 E h, PBST 5,. OD450/ 620nm,(= ( OD - OD ) / OD 100 %), 50 % pres1 mab 5 Balb/ c, Sp2/ 0 3, 90 % 88 %96 %, 50 % 56 %60 %. p res1 (21247) mab,4, 7 pres1 mab, 1 G5 2A6 3 H5 4D11 6F1 7B6 7 H11. D E252 mab,0. 52 mg mab,80 %. EL ISA mab 1 :4D11 7 H11 Ig G1, 5 mab Ig G2a ; 7B6,. EL ISA, mab.,mab ; mab mab HBV (1) mab Eppendoff,H EV mab E1 E2, Dane, PCR (1)., Dane. PCR, Dane. (2) Dane ( ) EL ISA 7 pres1mab HBV (Dane ). ( mab 1 G5 2A6 3 H5 4D11 6F1 7B6 7 H11 Ig G2a Ig G2a Ig G2a Ig G1 Ig G2a Ig G2a Ig G1

224 () 2008 1 HBV Fig. 1 Analysis of virus2neutralizing ability of HBV spe2 cific antibody in vitro 1. 2000DL Marker ;2. Dane paricle ;3. H EV mab E1 ; 4. H EV mab E2 ; 5.

2 The binding activity of pres1mabs to native HBV antigens Positive control : HBsAb 2), 7 pres1mab HB2 sab HBV., ( H EV) mabe1 E2. (20g/ ml),7 pres1 mab,mab 4D11 HBV. 2. 3 mab (1) mab HBV mab 4D11 HRP, EL ISA, 7 mab HRP2mAb 4D11,mAb HBV.

(4),3 H5 2A6 6F1 HBV (4,50 %), 4D11 1 G5 7B6 7 H11. pres1 (21247) B, mab 4D11 1 G5 7B6 7 H11,mAb 3 H5 6F1 2A6. 3 7 pres1 (21247) 3 pres1 pres1 4D11 HBV Fig.

3 224 () HBV Fig. 1 Analysis of virus2neutralizing ability of HBV spe2 cific antibody in vitro DL Marker ;2. Dane paricle ;3. H EV mab E1 ; 4. H EV mab E2 ; 5. anti2pres1 mab 1 G5 ; 6. anti2pres1 mab 2A6 ; 7. anti2pres1 mab 3 H5 ; 8. anti2pres1 mab 4D11 ;9. anti2pres1 mab 6F1 ; 10. anti2pres1 mab 7B6 ; 11. anti2pres1 mab. 7 H11 2 pres1 HBV Fig. 2 The binding activity of pres1mabs to native HBV antigens Positive control : HBsAb 2), 7 pres1mab HB2 sab HBV., ( H EV) mabe1 E2. (20g/ ml),7 pres1 mab,mab 4D11 HBV mab (1) mab HBV mab 4D11 HRP, EL ISA, 7 mab HRP2mAb 4D11,mAb HBV. 3,mAb 4D11 1 G5 7B6 7 H11 4D11 HBV, 50 %(3), 3 mab 3 H5 6F1 2A6 4D11 HBV. 4 pres1 mab 4D11 1 G5 7B6 7 H11 B. (2) 3 mab 3 H5 6F1 2A6, mab 6F1,7 mab. (4),3 H5 2A6 6F1 HBV (4,50 %), 4D11 1 G5 7B6 7 H11. pres1 (21247) B, mab 4D11 1 G5 7B6 7 H11,mAb 3 H5 6F1 2A pres1 (21247) 3 pres1 pres1 4D11 HBV Fig. 3 Competitive inhibition of anti2pres1 mabs against HRP24D11 binding with native HBV antigens 4 pres1 pres1 6F1 HBV Fig. 4 Competitive inhibition of anti2pres1 mabs against HRP26F1 binding with native HBV antigens

4 2 :HBV pres1 225 Ig G. EL ISA 7 HBV, :, mab 4D11 HBV,mAb 6F1. mab,pres1 [13 ].,HBV p res1 S2 HBV DNA,HbsAg,. pres1.,dna p res1 DNA ; HbeAg pres,pres HBV, pres1., pres1 [14 ]. EL ISA EL ISA,,7 p res1 (21247), B ;,mab HBV. mab 1 G5 4D11 7B6 7 H11 pres1 (21247) B, ; mab 2A6 3 H5 6F1 B. pres1 (21247) 2 B., mab 4D11 HBV, HRP2 mab 4D11 HBV ( ), pres1 HBV,, HBV.. HBV [7 ], pres , HbsAg, ; Hb2 sag [ 1 ], pres1 Fab Hep G2, HBs Fab. 7 mab Hep G2 ( ), mab, GST2 pres1 HBV.,,, HBV mab. : [1 ] Qiao M,Macnaughton T B, Gowans E J. Adsorption and penetration of hepatitis B virus in a nonpermissive cell line [J ]. Virology,1994,201 (2) : [2 ] De M S, Gong Z J,Suwandhi W,et al. Organ and species specificity of hepatitis B virus ( HBV) infection : a review of literature with a special regerence to preferential at2 tachment of HBV to human hepatocytes[j ]. J Viral Hep2 at,1997,4 (3) : [3 ] Heinz D,Peters M,Prange R,et al. Possible role of human interleukin26 and soluble interleukin26 receptor in hepati2 tis B virus infection[j ]. J Viral Hepat,2001,8 (3) : [4 ] De Falco S, Ruvoletto M G, Verdoliva A, et al. Cloning and expression of a novel hepatitis B virus2binding protein from Hep G2 cells[j ]. J Biol Chem,2001,276 (39) : [5 ] Budkowska A, Bedossa P, Groh F, et al. Fibronectin of human liver sinusoids binds hepatitis B virus : identifica2 tion by an anti2idiotypic antibody bearing the internal im2 age of the pre2s2 domain[j ]. J Virol,1995,69 (2) : [6 ] Hertogs K,Depla E,Crabbe T,et al. Spontaneous devel2 opment of anti2hepatitis B virus envelope (anti2idiotypic) antibodies in animals immunized with human liver en2 donexin II or with the F (ab ) 2 f ragment of anti2human liver endonexin II immunoglobulin G: evidence for a re2 ceptor2ligand2like relationship between small hepatitis B surface antigen and endonexin II[J ]. J Virol,1994,68 (3) : [7 ] Paran N, Geiger B, Shaaul Y. HBV infection of cell cul2 ture :evidence for multivalent and cooperative attachment [J ]. EMBO J,2001,20 (16) : [8 ],,,. [J ]., 2002,18 (6) : [9 ] Adler B, Faine S. A pomana serogroup2specific agglutina2 ting antigen in Leptospira,identified by monoclonal anti2 bodies[j ]. Pathology,1983,15 : [10 ] Harlow E,Lane D. Monoclonal antibodies[ M ]ΠΠHarlow E,Lane D editors. Antibodies :a laboratory mannal. New

5 226 () 2008 York : Cold Spring Harbor Laboratory Press, 1998 : [11 ],,,. [J ].,2003,19 (3) : [12 ] Sambrook J,Fristsch E F,Maniatis T. [ M ]ΠΠ,,,. 2. :,1998 : [13 ],,,. S1 [J ].,2007,23 (4) : [14 ] Tatsuji K,Akinori R,Akihiro M,et al. New enzyme im2 munoassay for detection of hepatitis B virus core antigen ( HBcAg) and relation between levels of HBcAg and HBV DNA[J ]. J Clin Microbiol,2003,41 : Isolation and Characteration of the Anti2native HBV pres1 Monoclonal Antibodies GU Ying,ZHU Zi2heng,L I Shao2wei,ZHAN G J un,xia Ning2shao 3 (National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences,Xiamen University,Xiamen ,China) Abstract : The Balb/ c mice were immunized with native HBV antigen (Dane particles and tubular particles) and purified recombi2 nant p rotein GST2preS1. Seven mabs anti2hbv pres1 mabs were prepared by hybridoma technique,and the relative p roperties of the mabs were identified by EL ISA and immu2capture PCR. Of these mabs,mab 4D11 and 7 H11 were Ig G1,the others were Ig G2a. The isotype of mab 7B6 light chains were lamda ( ),the others were kappa ( ). All of these mabs could recognize native HBV anti2 gens (Dane particles and tubular particles),f uther study shows that they could recognize two different B cell epitopes of pres1 (212 47),one recognized by mab 4D11,1 G5,7B6 and 7 H11,the other is recognized by mab 3 H5,6F1 and 2A6. The mabsabilities of recognizing the native HBV antigen show use of developing regent to test HBV pres1,and assisting in the design of neutralizing anti2 gen. Key words : monoclonal antibody ;hepatitis B virus ;pres1 ;B cell epitope

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