HBsAg, HBsAg. Tris - HCl, 200 mmol/ L NaCl, ph ml Amp 100 mg/ L, 4-5 h, [pqe - scfv] Kana 25 mg/ L 2 YT,37, 111 pqe - scfv

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1 Chinese Journal of Pathophysiology 2002,18 (9) : [ ] (2002) HBsAg 3 1, 2, 1, 2, 2, 2, 2, 1 ( 1, ; 2 458, ) [ ] :6 His, :,, 3 ;, :, ( ) % ; ( ) 10 7 L/ mol :,, N [], ; ; ; [] R [] A, 112 scfv N, RGS - His TM QIAGEN HBsAg HBsAg [1 ] His (hepatitis B surface antigen, HBsAg) pres2 - Trap TM Amersham - Pharma2 Fab [2 ], cia (single - chain antibody fragment, scfv), 113 Buffer A : 6 mol/ L GuHCl (8 mol/ L, Urea), 20 mmol/ L Na 2 HPO 4, 015 mol/ L NaCl, 10 HBsAg, mmol/ L imidazole, ph716 ; Buffer B : 6 mol/ L GuHCl, (8 mol/ L Urea), 20 mmol/ L Na 2 HPO 4, 0. 5 mol/ L Na2 3 Cl, 500 mmol/ L imidazole, ph7. 6 ; Buffer C : 10,, mmol/ L Tris - HCl, 1 mmol/ L EDTA, 0. 1 mmol/ L (affinity constant, K A ) GSH, 1 mmol/ L GSSG ph8. 0 ; Buffer D : 50 mmol/ L, Tris - HCl, 200 mmol/ L NaCl, ph ml Amp 100 mg/ L, 1 Kana 25 mg/ L 2 YT,37, 111 pqe - scfv YT,,Amp N 6 His 37 2 h A , IPTG HBsAg, ( , isopropyl - 1 T5 M15 [ prep4 ] QIAGEN - thio - - galactopyranoside) 1 mmol/ L,,pQE - scfv M15 [ prep4 ] M h, [pqe - scfv] 212 [3 ] : [ ] [ ] [ ] ( ) Tel : ; E - mail net PBS,,, 10 mmol/ L- Buffer A,4, : 0145m, Buffer A

2 1070 His - Trap TM,10 Buffer A,5 Buffer B,, SDS - PAGE 213 [4,5 ] : g/ L, Buffer C,Buffer C 4 mol/ L 0 mol/ L,:4 mol/ L 1 mol/ L 100 mmol/ L 10 mmol/ L 1 mmol/ L 0 mol/ L : 011 g/ L, Buffer D, Buffer D 3 mol/ L 0 mol/ L,,() :3 mol/ L 2 mol/ L 1 mol/ L 015 mol/ L 0 mol/ L 1 mol/ L 200mol/ L (oxidized form of glutathione, GSSG) 250 mmol/ L (L - Arg) : 6 mol/ L Buffer A,,10 Buffer A,,Buffer B 214 [6 ] ELISA HBsAg, RGS - His TM antibody,hrp Fig 1 SDS - PAGE analysis of the target protein produced in recom2 IgG, TMB, 450 binant M15 [pqe - scfv]. nm/ 630 nm Lane 1 : protein molecular markers ; Lane 2 : M15 [ pqe scfv] uninduced ; Lane 3 : M15[pQE - scfv] induced ; Lane HBsAg - Sepharose CL 4B, 011 mol/ L 4 : supernatant of the bacteria lysate ; Lane 5 : pellet of the PB (ph8. 0), 011 mol/ L Glycine - HCl (ph4. 0),Tris ph [7 ] 4 3 mg/ L 115 mg/ L 0175 mg/ L mg/ L HBsAg ELISA ; 10-7 mol/ L, , 256, ; RGS - His TM antibody HRP IgG, TMB,450 nm/ 630 nm g S,,3, K A = ( n - 1) / ( nab - Ab), Ab Ab Ag Ag, (mol/ L), n = Ag/ Ag n = 2, 3 K A, n = ) %, K A, n = 8 1 K A, 6 K A ( P < 0101) ; 217 Bradford [8 ], M15[pQE - scfv]iptg, 3112 %(1) ;,SDS - PAGE, 93 %,,Triton X bacteria lysate ; Lane 6 : lysed inclusion body ; Lane 7 : puri2 fied target protein by IMAC. 1 SDS - PAGE M15[ pqe - scfv] 2,, (2) : ( P < 0101, ANOVA ) ( ) A 450/ 630 U/g, (61108 ( P < 0101),

3 1071,,, ( P < 0101),, :10 mmol/ L, ( P < 0101), Fig 2 Dependence of the protein recovery and relative specific bioactivity on the methods of refolding. gx s. n = 6. 3 P < vs protein recovery of urea dialysis ; # P < vs specific bioactivity of urea dialysis. 2, 10 mmol/ L ( ), ( ) %, ( ) A 450/ 630 U/g,, ( P < 0101) 4 HBsAg - Sepharose CL 4B,,, 4, 4,, 6 K A (1),, ( ELISA, K A 1), ELISA 150 ng75 ng ng ng (8715 ng/ ng/ ),HRP HBsAg,,K = ( n - 1) / 2 ( nab - Ab) 1, ( ) 10 7 L/ mol, ( ) 10 8 L/ mol, ( P < 0101), Fig 3 Dependence of the protein recovery and relative specific bioactivity on the concentration of urea in the last refolding buffer. gx s. n = 6. 3 P < 0101 vs protein recovery of 0 mmol/ L urea ; # P < 0101 vs specific bioactivity of 0 mmol/ L urea. 3 3, (3) Fig 4 ELISA curves of refolded scfv to different amount of coated 1 mol/ L 0 mol/ L, antigen( gx s. n = 3). ( ) % ( ELISA 2152) %

4 ( hscfv) IgG ( migg) K A Tab 1 K A s calculated from the ELISA curves of human scfv and mouse IgG against HBsAg Antibody K n = 2 (10 7 L mol - 1 ) K n = 4 (10 7 L mol - 1 ) K n = 8 (10 7 L mol - 1 ) gx s (10 7 L mol - 1 ) HscFv MIgG t test P < K n = 2 is the collection of K A s calculated from every 2 curves differed in amount of coated antigen by 2 - fold. Analogized from this, K n = 4 and K n = 8 were calculated and listed above., mg/ L HBsAg : HBsAg B N 12, Fd,pComb3H,,, ;, HBsAg Fab ; PCR scfv DNA HB2,, sag HBsAg [11 ],, IgG 1-3,, ( ) 10 7 L/ mol, 1/ 16,, [9 ] [ ] N, C, [1 ] Kuttner G, Kramer A, Schmidtke G, et al. Characterization C [10 ] of neutralizing anti - per - S1 and anti - pre - S2 (HBV) mon2, oclonal antibodies and their fragments [ J ]. Mol Immunol, HBsAg,N 1999, 36 (10) : , [2],,,. - HBsAg Fab [J ]., 1999, 19 (6) : ,, [3 ] Wingfield PT, Palmer I, Liang SM. Folding and purification [A ]. In : Current Protocols in Protein science [M]. Vol 1.,, New York : John Wiley and Sons, Inc, (5) :1-27. [4 ] Kurucz I, Titus JA, Jost CR,et al. Correct disulfide pairing, and efficient refolding of detergent solubilized single - chain Fv GSSGArg, protein from bacterial inclusion bodies [ J ]. Mol Immunol, 1995, 32 (17-18) : D [5 ] Tsumoto K, Shinoki K, Kondo H, et al. Highly efficient re2 [5 ] HBsAg,( ) %, duction of oxidizing reagent - application to a human single - chain Fv fragment [J ]. J Immunol Methods, 1998, 219 (1 -, 2) : , [6 ] Sanchez L, Ayala M, Freyre F,et al. High cytoplasmic ex2,, Sanchez [6 ] IL - 2 HBsAg,,,5-6 of insoluble ( inclusion body) proteins from Escherichia coli covery of functional single - chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled intro2 pression in E. coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B sur2 face antigen [J ]. J Biotechnol, 1999, 72 (1-2) :

5 1073 [7 ] Beatty JD, Baetty BG, Vlahos WG. Measurement of mono2 clonal antibody affinity by non - competitive enzyme im2 munoassay [J ]. J Immunol Methods, 1987, 100 (1-2) : [8 ] Ausuble F, Brent R, Kingston RE, et al. Using the Bradford method to determine protein concentration [ A ]. In : Current Protocols in Molecular biology[ M]. Vol 2. New Yrok : John Wiley and Sons, Inc, [9 ] Sassenfeld HM. Engineering proteins for purification [ J ]. Trends Biotechnol, 1990, 8 (4) : [10 ] Passafiume M, Normand BV, Riottot MM, et al. Sequence analysis of a monoclonal antibody specific for the pres2 region of hepatitis B surface antigen, and the cloning, expression and characterization of its single - chain Fv construction[j ]. FEBS Lett, 1998, 441 (3) : [11 ],,,. HBsAg Fab [J ]., 2001, 17 (4) : Purification of an anti - HBsAg scfv and measurement of its affinity constant XIONG Sheng 1, REN Xiang - rong 2, YAN Xing 1,TANG Yong - hong 2, ZHENG Ye - hua 2, SU Kuan - yuan 2, YU Zhou - yao 2, YAO Ru - hua 1 ( 1 Department Biotechnology, South China University of Technology, Guangzhou , China ; 2 Center of Infections Diseases, the 458 th Hospital of PLA, Guangzhou , China) [ ABSTRACT] AIM : To purify and refold the inclusion body of a human anti - HBsAg scfv with a 6 His tag, and to determine the affinity constant of the purified recombinant product. METHODS : Solubilizing in buffers containing urea or guanidine hydrochloride ( GuHCl), the inclusion body was purified by IMAC, and then refolded by dialysis a2 gainst urea or GuHCl, at the same time, Ni 2 + charged chelate column was utilized for in situ refolding. The affinity con2 stant of the refolded scfv, polished by immune - affinity chromatography, was determined by non - competitive ELISA. RESUL TS : The refolded scfv with highest specific bioactivity was produced by dialysis against GuHCl. Under this con2 dition, the recovery of target protein reached ( ) %. The affinity constant of the polished scfv was confirmed to be ( ) 10 7 L/ mol. CONCL USION : The inclusion body studied in this paper can be refolded efficiently under optimal dialysis condition in vitro. The antigen - binding property of this recombinant scfv is not affected by the purification tag fused to the N terminal of the protein. [ KEY WORDS] Hepatitis B ; Antibodies ; Inclusion ; Antibody affinity ,89 800, 4 000,,,,, 635 (), 138 (),168 (),322 : 55 ; :100053: : : ; :