35 3 12 9 JOURNAL OF NANJING NORMAL UNIVERSITY Natural Science Edition Vol 35 No 3 Sept 12 pet28a + CD 210046 Rituximab VL VH CH3 CD overlap PCR Rituximab VL VH Linker ScFv ScFv IgG1 CH3 IgG1 cdna pet28a + IPTG SDS-PAGE Western blot CD 41 88 1 0 mmol /L IPTG 24 h 25% IgG1 CD CD B CD R394 4 A 1001-4616 12 03-0074-07 Construction and Expression of Anti-CD Minibody in pet28a + of Prokaryotic Expression System Li Dongxia Yang Zhen Pan Shaokun Zhou Nannan Song Fei Cao Xiangrong Abstract Objective Jiangsu Key Laboratory of Molecular and Medical Biotechnology School of Life Sciences Nanjing Normal University Nanjing 210046 China The research aimed to construct anti-cd containing VL VH and CH3 and to explore the best inducing condition of its soluble protein expression Method The recombinant cdna was produced by overlap PCR A short peptide linker was used to join VL and VH The human IgG1 hinge region was used to join VH and CH3 The recombinant gene was subcloned into the expression vector of pet28a and expressed in E coli BL21 Result SDS-PAGE and Western blot analysis showed that the recombinant protein was about 41 88 and the optimized soluble protein expression condition of was 1 0 mmol /L IPTG for 24 h at the inclusion bodies were found in the precipitation after sonication The soluble protein was further purified Ni-NTA affinity chromatography the yield up to 25% the highly purity recombinant protein was obtained after a series of steps including inclusion bodies cell lysis denaturation purfication and renaturation Western blot proved that the recombinant protein anti-cd had immunological reactive ability against rabbit anti-human IgG and can specific binding CD antigen The successful expressed of in the prokaryotic expression system was helpful for the future study of its biological function and target therapy to the B lymphoid leukemia and B lymphoma Key words CD prokaryotic expression E coli Non-hodgkin s lymphoma NHL B NHL 12-04-11 J110 E-mail caoxiangrong@ ninu edu cn 74
pet28a + CD 1 3 ~ 4 NHL 85% ~ % B 2 CD B B B 3-4 CD 5 CD B Rituximab C2B8 CD IgG1 CHO 6 1997 FDA C2B8 Minibody IgG VL VH CH3 Linker VL-VH-CH3 Fc ADCC ScFv 7 Rituximab CD B 1 1 1 DH5α puc19 CD cdna pdc316-atf BL21 pet28a + T4 DNA Pyrobest DNA Polymerase DNA marker Taq TaKaRa BioMI- GA His GE Healthcare IgG1 1 2 1 2 1 微抗体 ( ) cdna 的合成 pet28a VL VH CH3 1 pdc316 - ATF SalⅠ + EcoRⅠ 717 bp 28 bp 2 717 bp AT001 /AT004 CD cdna VL 5 028 bp AT002 /AT005 AT003 /AT006 CD cdna CD cdna VH CH3 VL VH CH3 3 VL VH AT001 /AT005 ScFv ScFv ScFv CH3 AT010 /AT011 1 147 bp Rituximab cdna CDMN 1 2 2 表达载体的构建 XbaⅠ + Hind Ⅲ PCR CDMN puc19 T4 DNA DH5α LB Amp + PCR puc19-cdmn NdeⅠ Hind Ⅲ pet28a + CD puc19-cdmn T4 DNA Kan + pet28a + - CDMN 1 2 3 重组基因的诱导表达及 SDS-PAGE 分析 pet28a + - CDMN BL21 Kan + 1% OD = 0 6 ~ 1 0 IPTG 3 2 IPTG 0 mmol /L 0 5 mmol /L 1 0 mmol /L 1 5 mmol /L 12 h 24 h h 16 25 75
35 3 12 SDS-PAGE IPTG AT001 AT002 AT003 AT004 AT005 AT006 AT010 AT011 1 2 4 蛋白的纯化和变复性 1 Table1 PCR Primers for the PCR 5'-CTAGTCTAGAAGATCTCAAATTGTTCTCTCCCAGTCT-3' 5'-GGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGGTACAACTGCAGCA-3' 5'-GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAGGGCAGCCCCGAGAACCA-3' 5'-GGAGCCGCCGCCGCCAGAACCACCACCACCAGTACGTTTGATTTCCAGCTT-3' 5'-GGTGCTGGGCACGGTGGGCATGTGTGAGTTTTGTCGGAGGCTGCAGAGACGG-3' 5'-CCCAAGCTTGCATGCTTACTATTTACCCGGAGACAGGGAGA-3' 5'-CTAGTCTAGACATATGCAAATTGTTCTCTCCCAGTCT-3' 5'-CCCAAGCTTAGATCTTTACTATTTACCCGGAGACAGGGAGA-3' 0 ml mmol /L Tris-cl ph = 8 0 174 μg /ml PMSF 13 000 rpm 4 6 min - PBS 6 000 rpm min 10 ml 2 mol /L 15 μl TritonX - 100 8 min ~ 10 min 6 000 rpm 4 min 5 4 mol /L 6 mol /L 8 mol /L 15 μl TritonX - 100 DTT 1 mmol /L 4 30 h 6 000 rpm 4 min 8 mmol /L I 4 30 h 6 000 rpm 4 min II 4 30 h 6 000 rpm 4 min III 4 3 5 h 6 000 rpm 4 min IV 4 6 000 rpm 4 min V 4 3 h - 1 2 5 Western blotting 鉴定重组蛋白 的抗原性 99 5 min SDS-PAGE 2 V min PVDF 1 h TBST 3 10 min IgG1 2 h TBST 3 10 min TMB 2 2 1 Minibody Overlap PCR pdc316-atf 717 bp 5 028 bp cdna 717 bp AT001 /AT004 cdna VL 5 028 bp AT002 /AT005 AT003 /AT006 cdna VH CH3 VL VH CH3 3 1 VL VH AT001 /AT005 ScFv ScFv CH3 AT010 /AT011 1 147 bp Rituximab cdna CDMN 2 M VL VL VL VH VH CH3 CH3 2 000 M 1 2 0 bp 2 bp 7 Fig1 Minibody 3 个功能区的 PCR 结果图 1 VL VH 和 CH3 基因扩增 The products of VL VH and CH3 by PCR amplification M DNA marker DL00; 1 1#; 2 2# 图 2 Overlap PCR 扩增 Fig2 The products of by overlap PCR amplification 76
pet28a + CD VL VH CH3 linker hinge Fig3 图 3 2 2 Minibody 结构图 Construction of CDMN PCR puc19 T4 9# 13# 4 9# 13# 1 128 bp 1 5 9# puc19-cdmn 9# puc19-cdmn 9 # pet28a + T4 Kan + pet28a + - CDMN 6 7 M 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 5 000 2 0 puc19-cdmn puc19 2 Fig4 M DNA marker DL100;0 puc19;1-16 puc19-cdmn 1-16# 图 4 菌液电泳初筛 puc19-cdmn 重组质粒 Identification of the recombinant puc19-cdmn by DNA fragments electrophoresis M 1 2 3 4 1 M 5 000 2 0 CDMN CDMN 2 000 1 0 2 M DNA marker DL100;1 puc19-cdmn 9#;2 puc19-cdmn 9#/Nde Ⅰ+BglⅡ;3 puc19-cdmn 13#;4 puc19-cdmn13#/ndeⅠ+bglⅡ 图 5 puc19-cdmn 酶切鉴定 Fig5 Enzyme digestion of the puc19-cdmn M DNA marker DL00; 1 pet28a(+)-cdmn 4#/ XbaⅠ+HindⅢ 图 6 pet28a(+)-cdmn 酶切鉴定 Fig6 Enzyme digestion of the pet28a(+)-cdmn 2 3 pet28a + - CDMN IPTG 42 florign IPTG Kan 1 0 mmol /L 1 0 mmol /L IPTG IPTG 1 0 mmol /L 24h 8 9 2 4 Fig7 IPTG 1 0 mmol /L 图 7 pet28a(+)-cdmn 6 426 bp HindIII(174) His-CDMN lacl NdIe(1296) XbIa(1393) 携带微抗体基因的表达载体 The expression vector harboring gene of 77
35 3 12 24 h 0 ml SDS- PAGE 0 μl SDS-PAGE 6# 7# 25% 63 33% 10 0 μl SDS-PAGE 96% 11 M 0 1 2 3 4 5 6 7 8 9 10 11 M 0 1 2 3 4 5 6 7 8 9 10 11 M Marker; 0 Ecoli 上清 ; 1 pet28α(+) 上清 ; 2 未诱导 pet28α(+)- CDMN 上清 ; 3-5 16 10 mmol/l IPTG 诱导 12 h 24 h h 上清 ; 6-8 10 mmol/l IPTG 诱导 12 h 24 h h 上清 ; 9-11 25 10 mmol/l IPTG 诱导 12 h 24 h h 上清图 8 不同温度 10 mmol/l IPTG 诱导不同时间的可溶性蛋白进行 12%SDS-PAGE 电泳 Fig8 12% SDS-PAGE analysis of soluble protein induced at 10 mmol/l IPTG, different temperature and different time M 0 1 2 3 4 5 6 7 8 9 10 11 M 1 2 3 4 5 6 7 8 9 M Marker; 1: pet28α(+) 上清 ; 2-3 10 mmol/l IPTG 诱导 24 h 上清 1 3; 4 Binding Buffer 洗脱液洗脱 1 号管 ; 5-9 Elution Buffer 洗脱液洗脱 1 2 3 4 5 图 10 镍柱纯化 12%SDS-PAGE Fig10 12% SDS-PAGE analysis of the purified through Ni + colume Fig11 2 5 Western blotting M Marker; 1-3 2 mol/l 尿素洗涤溶解 3 次上清 ; 4-5 4 mol/l 尿素洗涤溶解 2 次上清 ; 6 6 mol/l 尿素洗涤溶解上清 ; 7 8 mol/l 尿素重悬溶解 24 h 上清 ; 8 透析液 1 透析复性上清 ; 9 透析液 2 透析复性上清 ; 10 透析液 3 透析复性上清 ; 11 透析液 4 透析复性上清 ; 12 透析液 5 透析复性, 蛋白浓缩上清 图 11 Minibody 变复性 12%SDS-PAGE 12% SDS-PAGE analysis of after denaturation and renaturation M Marker; 0 Ecoli 沉淀 ; 1 pet28α(+) 沉淀 ; 2 未诱导 pet28α(+)- CDMN 沉淀 ; 3-5 16 10 mmol/l IPTG 诱导 12 h 24 h h 沉淀 ; 6-8 10 mmol/l IPTG 诱导 12 h 24 h h 沉淀 ; 9-11 25 10 mmol/l IPTG 诱导 12 h 24 h h 沉淀图 9 不同温度 10 mmol/l IPTG 诱导不同时间的包涵体进行 12%SDS-PAGE 电泳 Fig9 12% SDS-PAGE analysis of inclusion bodies at 10 mmol/l IPTG, different temperature and different time SDS-PAGE PVDF IgG1 TMB 42 Jurkat NAMALWA SDS-PAGE PVDF IgG1 35 3 CD 78
pet28a + CD 6 5 4 3 2 1 M 2 1 M CD A)a b GAPDH 5 4 3 2 1 M M 1 2 B)a b GAPDH A) 纯化的重组蛋白的抗原性和抗体活性检测 am Marker; 1 pet28α(+) 上清 0 mmol/l IPTG 诱导 24 h; 2 pet28α(+)-cdmn 上清 0 mmol/l IPTG 诱导 24 h; 3 pet28α(+)-cdmn 上清 10 mmol/l IPTG 诱导 24 h; 4-6 纯化重组蛋白 1 2 3; bm Marker; 1 Jurkat cell (CD-); 2 NAMALWA cell(cd+) B) 变复性重组蛋白的抗原性和抗体活性检测 am Marker; 1 pet28α(+) 沉淀 0 mmol/l IPTG 诱导 24 h; 2 pet28α(+)-cdmn 沉淀 0 mmol/l IPTG 诱导 24 h; 3 pet28α(+)-cdmn 沉淀 10 mmol/l IPTG 诱导 24 h; 4-5 变复性重组蛋白 Fig12 图 12 Western blotting 检测重组蛋白的抗原性和抗体活性 Western blotting analysis to show the specific binding of the protein NHL 8 B-ALL B B-NHL 9 NHL 10 Rituximab VL VH CH3 Linker IgG1 3 CH3 AD- CC Linker 4 1 15 9 H a - VL VH CH3 ADCC GlySer4 3 pet28a 6 His-Tag 9 IPTG IPTG 1 0 mmol /L 24 h 643 μg /ml 1 μg / ml Western blot 79
35 3 12 CD B 1 Jemal A Siegel R Ward E et al Cancer statistics J CA Cancer J Clin 08 58 2 71-96 2 CD J 05 25 7 34-39 3 Riley J K Sliwkowski M X CD a gene in search of a function J Semin Oncol 00 6 Suppl 12 17-24 4 Du J Wang H Zhong C et al Structural basis for recognition of CD by therapeutic antibody Rituximab J J Biol Chem 07 282 15 073-15 080 5 CD -Rituximab J 04 13 2 123-125 6 John R G Steven S H Jeffrey T R et al An afucosylated anti-cd monoclonal antibody with greater antibody-dependent cellular cytotoxicity and B-cell depletion and lower complement-dependent cytotoxicity than rituximab J Molecular Immunology 12 3 134-141 7 Hu S Z Lousise S Andrew R et al Minibody A novel engineered anti-carcinoembryonic antigen antibody fragment single-chain Fv-CH3 which exhibits rapid high-level targeting of xenografts J Cancer Res 1996 56 13 3 055-3 061 8 J 10 21 4 221-223 9 CD19 J 05 21 5 686-691 10 Verma R Boleti E George A J Antibody engineering comparison of bacterial yeast insect and mammalian expression systems J J Immunol Methods 1998 216 1 /2 165-181 80