An HBsAg Binding Protein Expressed in Pichia pastoris Can be Used as an HBV Vaccine Adjuvant

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology (11) : ,, 3 (, ) (HBsAg binding protein, SBP) HBsAg,., SBP., SBP, ( ELISA) (SPF) SBP, HBsAg,. SBP,SBP, HBsAg, SBP.,SBP,. ; ; Q78 ;Q An HBsAg Binding Protein Expressed in Pichia pastoris Can be Used as an HBV Vaccine Adjuvant PANG Yun, GONGLi, ZHU Nai2Shuo 3 ( Department of Microbiology and Microbiological Engineering, School of Life Sciences, Fudan University, Shanghai , China) Abstract HBsAg binding protain (SBP) was found in our previous study, which can specifically interact with HBsAg and whose ability on enhancing the immuno2response of HBV vaccine was proved. Now, with the Pichia pastoris expressing system, we have developed a Pichia pastoris strain expressing SBP secretively. In this work, we purified SBP through ultra2filtration and affinity chromatography, obtained high purified SBP. Then with the methods of ELISA and SPF, we characterized the biological activity of SBP, proved its affinity to HBsAg and calculated the affinity constant between them. After injection of SBP as an adjuvant of HBV vaccine to mice, compared with control group, SBP enhancing group showed an significant increase of HBsAb, proved the enhancing ability of SBP in humoral immunity. This work showed SBP can be used as an adjuvant of HBV vaccine, and was significant in Hepatitis B prevention and cure. Key words HBsAg binding protein ; enhancement of HBV vaccine ; adjuvant [1 ], [2 4 ].,, 5 % 10 % [5 ].,, 3 2.,, : ; : (No , ) 863 (No. 2006AA02Z462) 3 Tel : ,E2mail : nzhu - com Received : May 8,2009 ; Accepted : July 28,2009 Supported by National Natural Science Foundation of China (No , ) and National High Technology Research and Development Program of China (863 Program,No. 2006AA02Z462) 3 Corresponding author Tel : ; E2mail : nzhu - com

2 11 : 1025, [6,7 ].,., (hepatitis B surface antigen, HBsAg), cdna cdna. cdna, 1 344, ( HBV surface antigen binding protein, SBP, GenBank Accession : AY570731)., pbv2202sbp, E. coli DH5 SBP. Western ELISA, SBP HBsAg [8 ] ; SBP, HBsAg, SBP [9 ]. SBP, ppicz A2SBP, GS115, SBP. SBP,, GS2pPIC2SBP. BMGY ( 1118 % KH 2 PO 4, 013 % K 2 HPO 4 3H 2 O, 1134 % ( yeast nitrogen base YNB ), % (biotin), 1 % (yeast extract), 2 % (peptone) 2 % ( WΠV), BMMY2E ( 1118 % KH 2 PO 4, 013 % K 2 HPO 4 3H 2 O, 1134 % YNB, %, 1 %, 2 %, 2 mmolπl EDTA 015 %, WΠV), ; YNB BD ; Millipore ; ProteinA GE Healthcare ; (casamino acid) Sigma ;96 ELISA ; CM5 GE ; ; ; HBV ; SBP ; HRP SBP SBP GS2pPIC2 SBP ppicz A GS2pPIC 10 ml BMGY, rπmin 24 h 100 ml BMGY, rπmin 24 h,, 30 ml BMMY2E, 50 gπml Amp 15 gπml Kan,, 24 h 300 l, 72 h,. SDS2PAGE (12 % ), SBP 72 h 0122 m ; 10 kd (Millipore) 5 ;, ph 710 PBS ; PBS 710 Protein A, 5 ml, 215 mlπmin ;, PBS 710, 215 mlπmin, UV280 ; ph molπl, mlπmin,, 10 1 ph 910 Tris2HCl. HPLC SBP,Bradford SBP SDS2PAGE(12 % ) 200 ma 2 h NC, SBP ( ) HRP ( ) Western. SDS2PAGE(12 % ), MALDI2TOF2TOF2MS ELISA SBP HBsAg SBP HBsAg, SBP HBsAg K aff SBP. Beatty [10 ], 4 SBP ( mgπl) HBsAg SBP, HBsAg SBP HBsAg. HBsAg, A 492

3 , SBP A 492 ( 50 %A 492 ) HBsAg. : K = ( n - 1) [ 2 ( nab - Ab) ] K aff, Ab Ab SBP Ag Ag, A 492 HBsAg (molπl), n = AgΠ Ag. 4 Ag, 6 K aff, 6 K aff ( biomolecular interaction analysis, BIA) (SPR),,, Π Π Π [11,12 ]. Biacore3000, SPR,.,. SBP, HBsAg, Biacore HBsAg : molπl, SBP SBP ph 712 PBS, Bradford, PBS(pH 712) 2 mgπ ml,. 4 6 BalBΠC, 4, 6,, (18 2) g,,sbp 1 1,SBP 1 3 SBP 1 9. Table 1, ph 712 PBS 1 ml SBP,,4,37 1 h, 100 l, Table 2. HBsAg ELISA, HBsAb,. Table 1 preparation of the HBV vaccine2sbp enhancement test Group SBP volume Π l PBS volume Π l SBP mass Π g Vaccine volume Π l HBsAg Mass Π g HBsAg :SBP (molar ratio) Vaccine control SBP SBP SBP Table 2 Schedule of injection and blood sampling in the HBV vaccine2sbp enhancement test tπd Operation Blood sampling SBP GS2pPIC2SBP GS2pPIC 72 h SDS2PAGE(12 % ),. Fig. 1, GS2pPIC2SBP 35 kd, GS2 ppic, SBP,,. 212 SBP 1L GS2pPIC2SBP 200 ml,, ph, ph 315 SBP, HPLC 95 %. Western SBP SBP ( Fig. 2),

11 : 1027 SBP 2 mgπl, 50 % A 492 Fig. 1 Analysis of SBP expression by SDS2PAGE The fermentation fluid was collected after 72 hours inducement and separated by 12 % SDS2PAGE.

1 : Low M r protein marker ; 2 : HBsAg 116 mgπl (0170 10-7 molπl) ; SBP 5 mgπl,50 % A 492 HBsAg 114 mgπl (0161 10-7 molπl) ; SBP 10 mgπl,50 %A 492 HBsAg 1115 mgπl (0150 10-7 molπl).

4 11 : 1027 SBP 2 mgπl, 50 % A 492 Fig. 1 Analysis of SBP expression by SDS2PAGE The fermentation fluid was collected after 72 hours inducement and separated by 12 % SDS2PAGE. After electrophoresis, the gel was silver stained and scanned. 1 : Low M r protein marker ; 2 : HBsAg 116 mgπl ( molπl) ; SBP 5 mgπl,50 % A 492 HBsAg 114 mgπl ( molπl) ; SBP 10 mgπl,50 %A 492 HBsAg 1115 mgπl ( molπl). : K = ( n - 1) [ 2 ( nab - Ab) ], 6 6 K aff, K aff LΠmol. Fermentation fluid of GS2pPIC, negative control ; 3, 4 : Fermentation fluid of GS2pPIC2SBP Fig. 3 Calculation of the affinity constant K aff between SBP and HBsAg by ELISA SBP was enveloped in four concentrations(1, 2, 5, 10 mgπl) and incubated with HBsAg Fig. 2 Identification of purified SBP by SDS2PAGE and Western blotting 5 g purified SBP were separated by 12 % SDS2PAGE and gels were stained by Coomassie brilliant blue method(lane 1) or performing a Western blot (lane 3). When performing Western blot, protein was transfered onto the NC membrane and the blots were probed with anti2sbp monocloning antibody and HRP conjuncted goat2anti2mouse antibodies. Lane 2 : Protein marker. 213 ELISA SBP HBsAg SBP HBsAg, Fig. 3. SBP A 492 ( 50 % A 492 ) HBsAg : SBP 1 mgπl,50 % A 492 HBsAg 512 mgπl ( molπl) ; in different dilutions. Samples were detected by anti2hbsag monocloning antibody. A 492 was recorded for binding curve plotting and K aff was calculated with the method indicated below 214 SBP HBsAg Biacore3000, SBP HBsAg, Fig. 41 HBsAg : molπl, HBsAg, K aff LΠmol. 215 SBP Fig. 5 HBsAb, SBP,, SBP. 36 d,,

5 Fig. 4 Calculation of the affinity constant K aff between SBP and HBsAg by SPF SBP was immobilized to the chip and different concentration of HBsAg solutions were detected. HBsAg concentrations : 01106, 01213, 01425, 0185, 117, 314 molπl. K aff was calculated by software automatically. RU : response unit Fig. 5 Enhancement of HBsAg vaccine function by SBP SBP was purified, dialyzed with PBS (ph 712) and diluted to 2 mgπml. Endotoxin was removed. Mice were divided into four groups and injected with protein solutions as indicated in Table 11 and blood sampling were performed every 12 days as Table 21 Titer of anti2hbsag antibody was analyzed by ELISA. (A) Curves of antibody titer after injection ; (B) Antibody titers of four groups after 36 days, 3 3 P < 0105, 3 P < 0101 Pichia SBP. 3 SBP HBsAg, cdna [8 ].,.. SBP, SBP,,,., SBP., [13 ]., SBP., SBP. SBP HBsAg

6 11 : 1029, ELISA SPF,, ;,, SBP HBV., SBP,., SBP,,, SBP., SBP.,,., SBP,,,,. ( References) [ 1 ] World Health Organization. Hepatitis B vaccine2making global progress. EPI Update 31, 1996, Geneva : World Health Organization [ 2 ] Beasley R P, Hwang LY, Lee G C Y, et al. Prevention of perinatally transmitted hepatitis B infections with hepatitis B immunoglobuline and hepatitis B vaccine [ J ]. Lancet, 1983, 2 (8359) : [ 3 ] Hadler S C, Francis D P, Maynard J E, et al. Long2term immunogenicity and efficacy of hepatitis B vaccine in homosexual men [J ]. N Engl J Med, 1986, 315 (4) : [ 4 ] Thoelen S, Van Damme P, Mathei C, et al. Safety and immunogenicity of hepatitis B vaccine formulated with a novel adjuvant system[j ]. Vaccine, 1998, 16(7) : [ 5 ] Ottone S, Nguyen X, Bazin J, et al. Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for in vitro diagnosis[j ]. Protein Expr Purif, 2007,56(2) : [ 6 ] Singh M, O Hagan D T. Recent advances in vaccine adjuvants[j ]. Pharm Res, 2002, 19(6) : [ 7 ] Aguilar J C, Rodriguez E G. Vaccine adjuvants revisited [ J ]. Vaccine, 2007, 25 (19) : [ 8 ],. [J ]. ( Chen Yuan2Yuan, Zhu Nai2Shuo. Screening, expression and characterization of a novel protein binding to hepatitis B surface antigen[j ]. Chin J Biochem Mol Biol), 2005, 21 (1) : [ 9 ],. (SBP) [J ]. ( ) (Li Hua2Qing, Zhu Nai2 Shuo. Preparation of the HBV surface antigen binding protein and the functional study[j ]. J Fudan Univ Nat Sci), 2007, 46(3) : [10 ] Beatty J D, Beatty B G, Vlahos W G. Measurement of monoclonal antibody affinity by non2competitive enzyme immunoassay [ J ]. J Immunol Methods,1987,100 (122) : [11 ] Trevino J, Calle A, Rodriguez2Frade J M, et al. Determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay[j ]. Talanta,2009, 78 (3) : [12 ],. [J ]. ( ) (Wang Hai2Ming, Qian Kai2Xian. Principle of surface plasmon resonance biosensor technology and its application in biomolecular interacting analysis[j ]. J Zhejiang Univ Eng Sci), 2003, 37(3) : [13 ] Macauley2Patrick S, Fazenda M L, McNeil B, et al. Heterologous protein production using the Pichia pastoris expression system [J ]. Yeast, 2005, 22(4) :

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