ISSN 100727626 CN 1123870ΠQ Chinese Journal of Biochemistry and Molecular Biology 2009 11 25 (11) :1024 1029,, 3 (, 200433) (HBsAg binding protein, SBP) HBsAg,., SBP., SBP, ( ELISA) (SPF) SBP, HBsAg,. SBP,SBP, HBsAg, SBP.,SBP,. ; ; Q78 ;Q939191 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 200433, 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.,, :2009205208 ; :2009207228 (No. 30571650, 30471512) 863 (No. 2006AA02Z462) 3 Tel : 021265641215,E2mail : nzhu - 1 @sina. com Received : May 8,2009 ; Accepted : July 28,2009 Supported by National Natural Science Foundation of China (No. 30571650, 30471512) and National High Technology Research and Development Program of China (863 Program,No. 2006AA02Z462) 3 Corresponding author Tel : 021265641215 ; E2mail : nzhu - 1 @sina. com
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,,. 1 111 GS2pPIC2SBP. BMGY ( 1118 % KH 2 PO 4, 013 % K 2 HPO 4 3H 2 O, 1134 % ( yeast nitrogen base YNB ), 4 10-5 % (biotin), 1 % (yeast extract), 2 % (peptone) 2 % ( WΠV), BMMY2E ( 1118 % KH 2 PO 4, 013 % K 2 HPO 4 3H 2 O, 1134 % YNB, 4 10-5 %, 1 %, 2 %, 2 mmolπl EDTA 015 %, WΠV), ; YNB BD ; Millipore ; ProteinA GE Healthcare ; (casamino acid) Sigma ;96 ELISA ; CM5 GE ; ; ; HBV ; SBP ; HRP. 112 11211 SBP SBP GS2pPIC2 SBP ppicz A GS2pPIC 10 ml BMGY,30 200 rπmin 24 h 100 ml BMGY,30 200 rπmin 24 h,, 30 ml BMMY2E, 50 gπml Amp 15 gπml Kan,, 24 h 300 l, 72 h,. SDS2PAGE (12 % ),. 11212 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 315 011 molπl, 110 210 mlπmin,, 10 1 ph 910 Tris2HCl. HPLC SBP,Bradford - 80. SBP SDS2PAGE(12 % ) 200 ma 2 h NC, SBP (1 6 000) HRP (1 5 000) Western. SDS2PAGE(12 % ), MALDI2TOF2TOF2MS. 11213 ELISA SBP HBsAg SBP HBsAg, SBP HBsAg K aff SBP. Beatty [10 ], 4 SBP (1 2 5 10 mgπl) HBsAg SBP, HBsAg SBP HBsAg. HBsAg, A 492
1026 25, 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. 11214 ( biomolecular interaction analysis, BIA) (SPR),,, Π Π Π [11,12 ]. Biacore3000, SPR,.,. SBP, HBsAg, Biacore3000 2. HBsAg : 01106 01213 01425 0185 117 314 molπl, 2. 11215 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 0 1000 0 1000 20 SBP 1 1 2018 97912 33 1000 20 1 1 SBP 1 3 6215 93715 100 1000 20 1 3 SBP 1 9 18715 81215 300 1000 20 1 9 Table 2 Schedule of injection and blood sampling in the HBV vaccine2sbp enhancement test tπd 0 12 24 36 48 Operation Blood sampling 2 211 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. After electrophoresis, the gel was silver stained and scanned. 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). : K = ( n - 1) [ 2 ( nab - Ab) ], 6 6 K aff, K aff 413 10 6 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 11213 SBP HBsAg, Fig. 3. SBP A 492 ( 50 % A 492 ) HBsAg : SBP 1 mgπl,50 % A 492 HBsAg 512 mgπl (2126 10-7 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 : 01106 01213 01425 0185 117 314 molπl, HBsAg, K aff 1111 10 6 LΠmol. 215 SBP Fig. 5 HBsAb, SBP,, SBP. 36 d,,
1028 25 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
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