Screening, Expression and characterization of a Novel Protein Binding to Hepatitis B Surface Antigen

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 21 (1) :53 60, (, ) 3, cdna ( hepatitis B surface antigen binding protein, HBsAg2BP) cdna, cdna, 1 344, ptriplex, E coli XL12Blue 44 kd Western ELISA,, HBsAg Hep G2, cdna,, R512162,Q78 Screening, Expression and characterization of a Novel Protein Binding to Hepatitis B Surface Antigen CHEN Yuan2Yuan,ZHU Nai2Shuo 3 ( School of Life Sciences, Fudan University, Shanghai , China) Abstract In order to study the functional proteins involved in the process of hepatitis B virus invasion to hepatocytes, a cdna clone coding a protein binding to hepatitis B surface antigen( HBsAg) was screened out from a human liver cdna expression phage library by immunoscreening DNA sequencing analysis showed that the cdna had an open reading frame encoding 344 amino acid residues without transmembrane domain and belonged to immunoglobulin superfamily This gene was inserted into expression vector ptriplex and the recombinant fusion protein of 44 kd was purified from E coli XL12blue The specific binding ability of the recombinant protein to HBsAg was verified by Western blotting and ELISA Flow cytometry ( FCM) analysis showed that purified recombinant protein could enhance the affinity of HBsAg to human hepatoma cells Hep G2 This finding suggested that hepatitis B surface antigen binding protein ( HBsAg2BP) might be a soluble co2 receptor mediating hepatitis B virus invasion to hepatocytes Key words liver cdna phage expression library, hepatitis B surface antigen( HBsAg), hepatitis B surface antigen binding protein( HBsAg2BP) (hepatitis B, HB), 315 : , : (hepatitis B virus, HBV) (No ) RGC [1 ], 112 [2 ] HBV DNA, Med2X 3 Tel : (021) ,E2mail edu cn Received : March 12,2004 ;Accepted :May 10,2004, [3 ],HBV Supported by National Natural Science Foundation of China (No ), RGC Program of Hong Kong and Med2X Foundation of Fudan University 3 Corresponding author Tel : (021) ,E2mail edu cn

2 54 21 [4 ], HBV 1 (HBsAg) 3 : (S ), 111 (M,pre2S2 + S), (L,preS1 + pres2 + S), ( (CA,USA) ; Hepatitis B surface Antigen Hep G2 ) (ay) FITC HBsAg, IgA [5 ], Biodesign (ME,USA) ; ( asiloglycoprotein receptor, ASGPR ) [6 ], IgG Rockland ( PA,USA) ; (fibronectin) [7 ] [8 ], 232, HBV2BP (HBV binding protein) [9 ], (annexin ) [10 ], H(apolipoprotein H) [11 ], (Dassel,Germany) ; EcoR Xba,hIL2 6 ( human interleukin Takara (, ) ; Taq DNA, 6) pres1, HBV dntps (, [12, ] ; ) ; IPTG Budkowska [13,14 ] (, ) ; MEM HBV (HBV2BF), HBV pre2s1 pre2s2 Gibco (N Y,USA),, 50 kd, (, ), 112 HBV2BF E coli XL12Blue,BM25 8 Clontech ; Hep G2, Hep G2, HBV HBV 113 HBV cdna,, cdna XL12,HBV Blue LB, h 10 mmolπl IPTG, h,, 1 % 45 min,tbst(10 mmolπl ( pre2s1,pre2s2, S ), Tris2HCl,pH 810, 150 mmolπl NaCl, 0105 %Tween220), cdna 3 ; HBsAg(017 gπml) 1 h,tbst 3 ; FITC cdna, cdna HBsAg ( ) 1 h,tbst, 344, 3 ; ( ) 115 h,, TBST 3 ; BCIPΠNBT ( hepatitis B surface antigen, 2 binding protein, HBsAg2BP) 114 cdna ptriplex, E1 coli XL12Blue 25 l H 2 O,, 5, HBsAg2BP Western l,, PCR ELISA,HBsAg2BP, cdna Clontech ;, cdna, cdna HBsAg2BP TriplEx ptriplex, : Hep G2 BM ml LB,31,150 HBV, rπmin, A HBV, 350 l 1 lambda cdna Clontech FAST BCIPΠNBT Buffered Substrate Tablet Sigma (MO,USA) ; S&S (100 mmolπl NaCl, 10 mmolπl MgSO 4 7H 2 O, 35

3 1 : 55 mmolπl Tris2HCl, ph 715, 0101 % ),4 118 HBsAg2BP 10 ml BM l 1 molπl [16 ], MgCl l 150 l, ( 15 l, Marker min, 400 l LB,31, l) SDS2PAGE( = 15 %), 225 rπmin 1 h, 1 10 l LBΠ, (017 gπml), FITC carbenicillin,37 HBsAg ( ),, EcoR Xba (cdna ( ), TBST 3 ),, BCIPΠNBT, ptrip2 HBsAg2B P ptrip2 HBsAg2B P ( g),5 %, GenBank (www1ncbi1nlm1nih1gov) ptrip2hbsag2b P 3 ; PNPP 30 min,3 molπl XL12Blue, 5 ml LB NaOH, 405 nm ( Amp 100 gπml), LB ( Amp 100 gπml),240 rπmin A , IPTG 1 mmolπl, 4 h 5 h, ml PBS, (A) Hep G2 + ml 25 l, 25 l 015 g HBsAg + FITC HBsAg (1 2 SDS2PAGE, 5 min, 500) ; (B) Hep G g HBsAg 015 g g 10 min, 10 l 15 %SDS2PAGE HBsAg2BP + FITC HBsAg 117 HBsAg2BP HBsAg2BP + FITC 116 HBsAg (1 500) XL12Blue, 40 ml 015 g HBsAg,4 4 ml 20 mmolπl Tris2HCl (ph 715), 40 l 10 mgπml FITC HBsAg (1 500), 30, 30 min 200 l 10 % Triton2 min,pbs 2, 015 ml PBS, X l 011 molπl PMSF,37 15 min 1, 1 min 115 ml,4, g 10 min,, 211 HBV cdna ( ) 750 l 20 mmolπl Tris2HCl cdna, g 5 min,, (independent clones) , ml 1 %SDS, %SDS2PAGE, ( pre2s1,pre2s2, S Walker [15 ],,PBS ,,, PEG ,,12 %SDS2PAGE, 212 cdna 2 h, HBsAg (250 ngπ ), BLAST h,tbst 3 ; FITC (ORF) HBsAg ( ), h,tbst 116 HBsAg2BP 3 ; ( ),37 1 h,tbst 1110 HBsAg2BP HBsAg FCM Hep G2,PBS 3, (1 500) ; (C) Hep G g HBsAg 110 g, 30 min, PBS 2, 015 ml 2 ),,

4 56 21, cdna 114 kb GenBank BLAST, (, Fig11) cdna, BM2518 Cre loxp ptriplex 3,, TriplEx cdna ptrip2 HBsAg2B P 3 ptriplex ( Fig12) EcoR Xba, 315 kb ptrip2 HBsAg2B P XL12Blue, 1 (ptriplex ) 112 kb ( cdna) mmolπl IPTG, 15 %SDS2PAGE ptrip2 HBsAg2B P 214 HBsAg2BP, 44 kd (Fig11), cdna ptriplex,, 44 kd ( Fig13), Fig 1 PCR amplification of target cdna and restriction analysis of recombinant ptriplex M DNAΠEcoT 14I marker ;1 Recombinant plasmid ; 2 Recombinant plasmid digested with EcoR and Xba ; 3 PCR product of positive phage Fig 3 SDS2PAGE analysis of the expression product M Protein molecular weight marker ;1 XL12blue control ; 2 XL12Blue containing recombinant ptriplex without IPTG induction ; 3, 4 XL12blue containing recombinant ptriplex induced by 1 mmolπl IPTGfor 4 hours and 5 hours,respectively Fig 2 Conversion of a recombinant TriplEx to ptriplex 215 HBsAg2BP IPTG XL12Blue, 12 %SDS2PAGE, (Fig14) HBsAg2BP, 12 % SDS2PAGE, 44kD (Fig15),, 0114 mgπml 216 IPTG, SDS2PAGE, HBsAg 213 ( ptrip2 HBsAg2B P) HBsAg2BP, FITC HBsAg, cdna IgG, bp, 344 ( Fig16), 38 kd,, pi 810,, HBsAg Western ( GenBank accession AY570731),IPTG 1 mmolπl,

5 1 : h, 5 h Fig 4 Existence of target protein in inclusion body M Protein molecular weight marker ; 1, 2 Inclusion body extracted from recombinant with IPTG induction ; 3 Supernatant of recombinant with IPTG induction ; 4 Inclusion body extracted from recombinant without IPTGinduction Fig 6 Western blotting of target protein M Prestained protein molecular weight ; 1 XL12Blue ; 2,3 Recombinant induced for 6 hours by 10 and 1 mmolπl IPTG, respectively ; 4,5 Recombinant induced for 4 5 hours by 10 and 1 mmolπl IPTG,respectively Fig 7 ELISA analysis of purified recombinant HBsAg2BP and HBsAg Fig 5 Purified HBsAg2BP M Protein molecular weight marker ; 1 Purified HBsAg2BP 217 FCM HBsAg Hep G2, HBsAg A, HBsAg2BP g B C,M , HBsAg Hep G2 ( Fig18 ), HBsAg2 BP HBsAg Hep G2 HBsAg2BP, 3 HBsAg, 3, Fig17,,, A 405, cdna HBsAg HBsAg2BP HBsAg 218 HBsAg2BP HBsAg ( HBsAg),, FCM, Hep G2,,

6 58 21 Fig 8 FCM analysis of the effect of purified protein on the binding ability of HBsAg to Hep G2 cells (A) Control ; (B) Purified protein 015 g ; (C) Purified protein 110 g M2 :positive ratio ; FL12H:represents first channel s fluorescence intensity, HBV, cdna TriplEx,, HBV TriplEx cdna, HBV cdna, ,,, cdna HBV, 1226 bp, 344 HBV, HBV,, cdna, [20 22 Cre loxp ] ptriplex, E1 coli, XL2Blue IPTG, (44 kd), HBsAg, Western, ELISA, HBsAg2BP HBsAg,, HBV HBsAg ( ) IgG [23,24 ],, HBV IgG Fc, HBV, HBV (FcRn), IgG, [25,26, HBV IgG ] FcRn 2, MHC2I, (HBsAg2BP) ph Fc IgG [27 ],, HuH27 ( HBV2negative), PLCΠPRFΠ5 ( HBV2positive,, HIV HBsAg), CD4 ( IgG,, CCR5ΠCXCR4 ) [17 ], FcRn IgG, hpvr (CD155) [18 ], HAVcr21 [19 ] [28 ] HBsAg2

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