H IV-1 gp160. Ana lysis and Expression of An ti-h IV-1 Id iotyp ic An tibody. H IV 21 gp 160. , 1) Fab. gp 160

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1 14 4 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy A ug 1998 H IV-1 gp160 1) 2) (, ) ) 1F7 H IV 21 gp 160 3B Fab, 1D Fd Fd, V H V Hg, 3B ϑ Κ Fab 3 3B H IV 21 gp 160, 3B ṡ 1D 3B s 1F7 gp 160 :,, Ana lysis and Expression of An ti-h IV-1 Id iotyp ic An tibody Zhao Yun2Feng W ang Q uan2l i D u Zh i2yan Sun Hong2Yan Yang J ing2geng W ang H ai2t ao M a L i2r en Y in Zhen (Institu te of T ransf usion M ed icine, B eij ing ) Abstract In o rder to classify the phagem id an tibodies and to purify them sim p ly, tw o hum an an ti2h IV 21 idio typ ic an tibodies w ere analyzed by restriction enzym atic m app ing and DNA se2 quencing, and so lub le exp ression vecto r w as con structed Phagem id 1D w as found on ly con2 tain ing a Fd segm en ṫ Bo th of the Fd segm en ts w ere the sam e T he V H gene w as derived from V H g subgroup, JH from HUM JH 6 T he D segm en t con tained D 2D fu sion T he ligh t chain of 3B did no t belong to any of ϑo r Κchain fam ilieṡ A fter recon stitu tion the Fd w ith o ther Κ ligh t chain s of an ti2hb sa g Fab, a new clone nam ed 3B s w as so rted ou t w h ich exh ib ited tigh ter b inding ab ility of gp 160 than to HB sa g So lub le an tibodies of 3B s and 1D w ere ex2 p ressed in the bacterial cu ltu re T he specificity of the an tibodies w as p roved by imm unob lo t assay Key words: Phage an t ibody lib ra ry, Sequencing, Exp ression ( Ig), 1), ), : , :

2 Ig Ig ϑ Κ EL ISA pcom b3 Fd g C Fd [ 1 ] H IV 21 1F7 ( [2 ]) gp 160 Fab Fd Xho g + Speg pb luescrip t SK (+ ) M 13m p 18 M 13m p 19, Sacg + Xbag L ife Science Seque2 nase V ersion 210 DNA Sequencing K it, 35 S2dA T P D upon, 32 P2dA T P 112 D NA 50 m l SB 2 m l XL 12B lue, 015 m l, 5 m in, h rgm in 10 m in 35 m l, 1g9 40% PEG g9 5 m o lgl (ph 7), 4 4 h rgm in 10 m in, 2 m l T E ( 10 Λl R nase), 20 m in (ph 810) 5 m in m o lgl (ph 512) h DNA 50 Λl 113, Speg + N heg g C XL 12B lue Fab 114 Fab 2 m l m l SB ( 50 Λggm l 20 mm o lggl M gc l2), A 600 = 012, IPT G 1 mm o lgl, 30 2 d rgm in 10 m in, rgm in 10 m in Fab 115 B io2r ad H um an H IV 21 N ovapath TM Imm unob lo t K iṫ 300 Λl Fab 3 m l, 30 m in, 10 m in 1g 100 1F7 ( [ 2 ], 5 m ggm l), 30 m in 1g1000 HR P2 IgM ( Sigm a ), 30 m in, DAB H IV-gp160 3B 1D, 3B Fd ( F ig 1A ) 1D Fd ( F ig 1B ) Xhog 378

3 1D 3B ( F ig 1C) N heg A pag, 1D, 3B 1D 600 bp ( F ig 1D ), 1D F ig 1 Restriction enzym atic m app ing of 3B and 1D (A )M app ing of 3B (B)M app ing of 1D (C) 11Phagem id 3B (D )M 1ΚDNA B steg 11Xhog + Speg digest 11Xhog + Speg digest 213B digest by Xhog 111D digest 21Sacg + Xbag digest 21Sacg + Xbag digest M 1ΚDNA EcoRg + H indg by N heg + A pag M 1123 bp ladder M 1123 bp ladder 31Phagem id 1D 213B digest 411D digest by Xhog by N heg + A pag 212 M 13m p 18 M 13m p 19 Xhog Speg, 3B 1D Fd pb luescrip t SK (+ ) Fd (F ig 2) Sequences of p ro tein s of imm uno logical in terest V H V Hg, D 2 D gene : DHQ 52 DM 1, D 2D J JH 6,, 3, 2 (111 Gly A la, 125 Ser A la) CDR 2 51bp, 17 3B bp ϑ Κ Β ( 50% 58 bp ) [ 3 ] 3 HB sa g Κ XL 12B lue V CSM 13 EL ISA L 36 (V L V Κg ), 3B s ( T ab le 1) 213 Speg + N heg Fd g, 379

4 F ig 2 DNA and am ino acid sequences of clone 3B and 1D 5, 3B s 1D gp 160, gp 41, p 24 ( F ig 3; 1, 2 3B s, 3 1D, 4 ) Table 1 EL ISA of reconstructed clone 3B s Phage antibody w ith gp160 A 490 w ith HB sa g clone 3B clone 3B s V CSM Λl of phage2antibody w as added to m icro titer p late w ells coated w ith 400 ng of gp160 and HB sa g T hen incu2 bated at 37 fo r 2 h A fter w ash ing th ree tim es w ith PBSg T and two tim es w ith H 2O 2, HRP2Conjugated rabbit anti2 M 13 (1g 1000) w as added and incubated fo r 1 h at 37 A fter w ashing w ith PBSgT and H 2O 2, bound antibodies w ere visualized using O PD F ig 3 Imm unoblo t assay of clone 3B s and 1D 3 PCR 3B V H V H g, 380

5 [ 4 K lasse H IV 21 IgG1 ] ϑ Κ, Fd H IV 2 [ 5 : gp 120 Κ ] Fab Κ 3B Fd, EL ISA EL ISA, Fab gp 41 p 24 pcom b3, Fd 1D,,W u Kabat [ 6 ] (CDR ), CDR s CDR 6 CDR s [ 7 ], DNA [ 8 BVO ] CDR, H 3,, H [ 9 ] L evi [ 10 H IV 21 V 3 H 3 ], 1D Fd H 3 References 1 Barbas C F, L erner R A Com binato rial imm unoglobulin libraries in phage Κ,M ethods A Comp anion tom ethod s in E nzy 2 m ology, 1991, 2 (2) : M uller S,W ang H T, Kaveri S V, Chattopadhyay S, Koh ler H Generation and specificity of monoclonal anti2idio typ ic an2 tibodies agninst hum an H IV 2specific antibodies J Imm unol 1991, 147: R ini J M, Sch ilize2gahm en U,W ilson IA Structural evidence fo r induced fit as a m echanism fo r antibody2antigen recog2 nition S ciencs, 1992, 255: Klasse P J, B lom berg J, P ipko rn R D ifferential IgG subclass response to ep itopes in transm em brane p ro tein of H IV 21 V iral imm unol, 1990, 3: 89 5 Koh ler H,M uller S, N ara P L D ecep tive imp rinting in the imm une response against H IV 21 Imm unol T od ay, 1994, 14 (10) : W u T T, Kabat E A A n analysis of the sequences of the variable regions of Bence Jones and m yelom a ligh t chains and their imp lications fo r antibody comp lem entarity J E xp M ed, 1970, 132: W ilson I A, Stanfield R İ A ntibody2antigen interaction Cu rr Op t S truct B iol, 1993, 3: H erron J N T h ree2dim ensional structure of a fluo rescein2fab comp lex crystallized in 22m ethyl22, 42pentanedio l P ro2 teins, 1989, 5: Sanz İ M ultip le m echanism s participate in the generation of diversity of hum an H 2chain CDR 3 regions J Imm unol, 1991, 147 (5) : L evim, Sallberg M, Ruden U, H erlyn D,M arhyam a H,W igzell H,M ark s J,W ah ren B A comp lem entarity2determ ining region synthetic pep tide acts as a m iniantibody and neutralizes hum an imm unodeficiency virus type I in v itro P roc N atl A cad S ci U SA, 1993, 90: