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1 生物工程学报 Chin J Biotech 2009, November 25; 25(11): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 医学与免疫生物技术 hk-fc 周同 1, 陶建军 1, 李林国 1, 侯永敏 2, 余龙 1 1, , : 为了延长人激肽释放酶 (hk) 的血清半衰期, 提高分泌蛋白的产率, 制备了重组激肽释放酶 -IgG1 Fc 融合蛋白 (hk'-fc) 采用 PCR 扩增 hk 基因和 IgG1 的 Fc 序列, 用鼠源信号肽序列替换 hk 基因原有的信号肽序列, 构建改良型融合蛋白 hk'-fc 以及天然型融合蛋白 hk-fc 的表达载体, 转染中国仓鼠卵巢细胞 (CHO) 细胞, 筛选稳定分泌融合蛋白的细胞株, 通过 Western blotting 鉴定信号肽改造效果, 利用 Protein A+G 亲合层析柱纯化融合蛋白, 酶学实验检测融合蛋白的体外活性 结果表明 : 成功构建了 pcdna-hk'-fc 以及 pcdna-hk-fc 重组表达载体 ; 获得了稳定表达融合蛋白的细胞株, 产量达 11 mg/l 以上 ; 信号肽改造后融合蛋白的分泌效率提高约 5~10 倍 ; 融合蛋白能水解其特异性的底物 S-2266, 具有生物学活性 本研究为进一步探讨融合蛋白的体内半衰期打下了坚实基础, 也为研制治疗脑梗塞疗效更好的第二代 hk 蛋白和其他药用蛋白的改良提供新的线索 : 激肽释放酶, hk'-fc 融合蛋白, 血清半衰期, 信号肽改造, 酶活性 Modification, expression and bioactivity analysis of hk-fc fusion protein Tong Zhou 1, Jianjun Tao 1, Linguo Li 1, Yongmin Hou 2, and Long Yu 1 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai , China 2 Techpool Bio-Pharma Co., Ltd., Guangzhou , China Abstract: To prolong serum half-life of human kallikrein (hk) and enhance its secretion rate, we modified hk gene and constructed a new form of recombinant hk protein (hk'-fc). We amplified hk gene and Fc sequence, replaced the signal peptide of hk gene with murine signal peptide, constructed native expression plasmid of pcdna-hk-fc and modified expression plasmid of pcdna-hk'-fc, then transfected to CHO cells respectively. After the stable cell lines were screened, we compared the secretion rate between native fusion protein and modified fusion protein, purified fusion protein through Protein A+G affinity chromatography column and investigated the bioactivity of fusion protein. The results showed that recombinant vectors encoding fusion protein hk-fc and hk'-fc were constructed successfully; CHO cell lines stably secreting fusion protein were obtained, the yield is higher than 11 mg/l; Secretion rate was enhanced by 5 10 times after the signal peptide of fusion protein was modified; Fusion protein has enzymatic activity in vitro. The above results could promote the following researches on serum half-life of the fusion protein and develop a new stroke medicine with better clinical efficacy. Keywords: kallikrein, hk'-fc fusion protein, serum half-life, signal peptide, bioactivity Received: July 10, 2009; Accepted: September 30, 2009 Corresponding author: Long Yu. Tel: ; Fax: ; Longyu@fudan.edu.cn

2 1698 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11 1(Human Kallikrein 1, hk) [1], [2-5] hk 43 kd, 238 [6] (Kininogen), (Kinin) [7], [8-10], hk hk, [11] hk,, hk,,,, IgG1 Fc,, : IL-2 TNF-α IFN-α [12-13] hk, hk Fc,,,, hk hk,, hk Coloma Ig κ (Ig κ-chain leader sequence) [14], Signal P [15-16] hk, hk, hk,, 1 材料与方法 1.1 主要试剂及抗体 Marker, Lipfectamine TM 2000 Invitrogen ; (BamH I Xho I EcoR I) T4 DNA Taq Pyrobest DL2000 DNA marker TaKaRa ; ; Tiangen ; NC ; HyQ SFMCHO CHO Hyclone ; (FCS) DMEM GIBCO ; hk ; IgG1 Fc-HRP ; G418 Sigma ; DNA Invitrogen ; S-2266 ; hk 1.2 载体与细胞 pcdna TM 3.1/myc-His(-) A Invitrogen ; pcmv-hk ; pmd18-t-fc ; CHO 1.3 实验引物 hk hk F hk R3, Fc Fc-F2 Fc-R 4 Ig maf hk IgF2 hk IgF3 hk IgF4, hk R hk 基因 改良型 hk 基因 (hk') 和 IgG1 Fc 片段的扩增 pcmv-hk, hk F hk R3 hk PCR : 94 o C 4 min; 94 o C 30 s, 62 o C 30 s, 72 o C 30 s, 35 ; 72 o C 5 min

3 : hk-fc 1699 表 1 实验所需引物的序列 Table 1 Primers used in the study Primer Sequence (5 3 ) hk F GTGACTCGAGACCATGGGGTTCCTGGTTCTGTGC (Xho I) hk R3 ATCTGAATTCGGAGTTCTCCGCTATGGTGTC (EcoR I) Fc-F2 GTGAGAATTCGGCGACGAGCC CAAATCTTGTGACAAA (EcoR I) Fc-R ATCTGGATCCTCATTTACCC GGGGACAGGGAG (BamH I) Ig maf CCAGGTTCCACTGGTGACATTGTGGGAGGCTGGG hk IgF2 TACTGCTGCTCTGGGTTCCAGGTTCCACTGGTG hk IgF3 CACACTCCTGCTATGGGTACTGCTGCTCTGGG hk IgF4 GTGACTCGAGACCATGGAGACAGACACACTCCTGCTATGGG (Xho I) The restriction enzyme sites for EcoR I (GAATTC), BamH I (GGATCC) and Xho I (CTCGAG) included in the PCR primes are indicated by underlining. pmd18-t-fc, Fc-F2 Fc-R IgG1 Fc, PCR : 94 o C 4 min; 94 o C 30 s, 58 o C 30 s, 72 o C 30 s, 35 ; 72 o C 4 min PCR hk' PCR, 4 PCR hk N PCR pcmv-hk, Ig maf hk R3, hk N 18 bp C ; PCR PCR, Ig F2 hk R3, 17 bp N ;, PCR PCR, hk N, Ig κ hk N 1.5 重组表达载体的构建和验证 Xho I EcoR I hk PCR pcdna 3.1,, pcdna-hk, DH5α,, BamH I EcoR I pcdna-hk Fc, pcdna-hk-fc PCR, DNA CHO pcdna-hk'-fc, hk' hk, pcdna-hk-fc 1.6 稳定细胞株的构建 CHO 10 % DMEM 24 h, CHO /ml 35 mm 0.5 h Lipfectamine h 24 h, 1: mm, G μg/ml 14 d 96 24, 1.7 融合蛋白的鉴定 Western blotting 10% HyQ SFMCHO 3 d,, g 5 min 80 μl, 20 μl 5 SDS loading buffer, 35 mm 400 μl 1 SDS loading buffer 95 o C 10 min, SDS-PAGE 80 V, 30 min, 120 V 2 h 4 o C 100 V 2.5 h (1:5000) (1:5000), ECL hk Fc d, Western blotting hk

4 1700 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11, Quantity One,, 1.8 信号肽改造效果的鉴定 h, CHO /ml h, 500 L 400 ng pcdna-hk-fc pcdna-hk'-fc DNA CHO 72 h, hk-fc hk'-fc /ml h, 2, 1.9 融合蛋白的纯化 72 h,, 2000 g 5 min, 1/10, PBS(pH 7.4) : 2, 0.2 mol/l Tris 1 ml 0.1 ml S-2266, 37 5 min, 0.05 ml,, 405 nm 2 min, 1 h 2: 2, 0.2 mol/l Tris 1 ml 0.05 ml 37 o C 5 min, 0.1 ml S-2266, 405 nm 2 min, 1 h 2 结果 2.1 hk 基因 Fc 序列以及 hk' 基因的克隆 pcmv-hk hk, hk 789 bp; pmd18-t-fc Fc IgG 1 Fc 699 bp ( 1) Amershan pharmacia Protein A+G, 12% SDS-PAGE Western blotting 1.10 融合蛋白酶活性分析 , 1, ml 0.2 mol/l Tris 2.0 ml,, (37±0.5) o C 5 min, 1 50% 0.2 ml, ml, (37±0.5) o C 15 min, ml, % 0.2 ml, 405 nm, 1, 2 3 A, A : PNA /ml =173.6 A T/1000, ; T ; 1000 /L /ml 图 1 hk 基因和 Fc 序列的扩增 Fig. 1 PCR product of hk gene and Fc sequence. M: DNA marker DL2000; 1: PCR product of Fc sequence; 2: PCR product of hk gene. hk' 4 PCR( 2), Ig κ hk N, hk' hk' 780 bp, ( 3)

5 : hk-fc 融合蛋白表达载体的构建与鉴定 EcoR I/Xho I hk pcdna 3.1, pcdna-hk, EcoR I/BamH I Fc, pcdna-hk, pcdna-hk-fc ( 4A) PCR ( 4B), pcdna-hk- Fc pcdna-hk'-fc pcdna-hk-fc 图 4 真核重组表达载体 pcdna-hk-fc 的酶切 (A) 和 PCR 鉴定 (B) Fig. 4 Restriction enzyme analysis (A) and PCR analysis (B) of pcdna-hk-fc. (A) M: DNA marker DL2000; 1: hk gene, pcdna-hk-fc digested with EcoR I/Xho I; 2: hk-fc sequence, pcdna-hk-fc digested with Xho I/BamH I; 3: Fc sequence, pcdna-hk-fc digested with EcoR I/BamH I. (B) M: DNA marker DL2000; 1: Fc sequence, pcdna-hk-fc amplified with primers Fc-F2 and Fc-R; 2: hk gene, pcdna-hk-fc amplified with primers hk F and hk R3; 3: hk-fc sequence, pcdna-hk-fc amplified with primers hk F and Fc-R. 图 2 逐步延伸 PCR 克隆 hk' 基因 Fig. 2 Cloning of hk' gene. 图 3 hk' 基因最终 PCR 产物 Fig. 3 Final PCR product of hk' gene. M: DNA marker DL2000; 1: hk' gene. 2.3 融合蛋白的表达和鉴定 Western blotting hk'-fc, hk ( 5A) Fc ( 5B) 4, CHO,, 66 kd 2.4 信号肽改造效果鉴定 pcdna-hk-fc pcdna-hk'-fc Western blotting ( 6A) μl, 20 μl hk-fc hk'-fc μl, 25 μl hk'-fc

6 1702 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11 图 5 hk 多抗 (A) 和 Fc 单抗 (B) 鉴定融合蛋白 Fig. 5 Identification of fusion protein by anti-hk polyclonal antibody (A) and anti-fc monoclonal antibody (B). (A) 1: stable cell lines; 2: supernate of stable cell lines; 3: CHO cells; 4: supernate of CHO cells. (B) 1: CHO cells; 2: supernate of CHO cells; 3: stable cell lines; 4: supernate of stable cell lines. 图 6 信号肽改造效果的瞬时转染 (A) 和稳定转染 (B) 鉴定 Fig. 6 Comparison of secretion rate by transient transfection (A) and stable transfection (B). (A) 1: supernate of pcdna-hk- Fc; 2: supernate of pcdna-hk'-fc; 3: pcdna-hk-fc cells; 4: pcdna-hk'-fc cells. (B) 1: supernate of T8; 2: supernate of Z97; 3: T8 cells; 4: Z97 cells. T8: stable cell line secreting hk-fc; Z97: stable cell line secreting hk'-fc. hk-fc, hk-fc T8 hk'-fc Z97, 12 L T8 3 L Z97, 12 L T8 3 L Z97, Z97 T8 ( 6B) 5~ 细胞稳定株的分泌量测定 Western blotting hk, 1 ng 3 ng 5 ng 7 ng 9 ng 11 ng 6,, 7 Z58 hk'-fc mg/l 图 7 细胞稳定株分泌融合蛋白的产量鉴定 Fig. 7 Yield determination of stable cell lines. 1: supernate sample; 2: 1 ng hk protein; 3: 3 ng hk protein; 4: 5 ng hk protein; 5: 7 ng hk protein; 6: 9 ng hk protein; 7: 11 ng hk protein. 2.6 融合蛋白的纯化与鉴定, PBS(pH 7.4), Protein A+G, Western blotting SDS-PAGE ( 8) : 66 kd, Western blotting hk'-fc, 66 kd,, 2.7 融合蛋白酶活性的测定 S-2266, hk'-fc 1.77 PNAU/mL

7 : hk-fc 1703 hk'-fc S-2266,, : 1 2 1,, ; 2,, 9, 1 2, , :, S-2266 图 8 纯化后融合蛋白的鉴定 Fig. 8 SDS-PAGE analysis of purified fusion protein. 1: purified fusion protein sample; 2: supernate sample before purification. 图 9 融合蛋白的酶活曲线 Fig. 9 Bioactivity analysis of fusion protein. 1: assay 1-fusion protein was added in reaction solution (buffer+s-2266); 2: assay 2-S-2266 was added in reaction solution (buffer+fusion protein). 3 讨论, hk, 30 min, T1/2β 156~197 min, hk hk, 24 h hk, IgG 2 Fab Fc IgG 21 d, Fc [17], IgG1 Fc,, (EPO) Fc, [17] Enbrel(TNFR-Fc), TNF Fc,, hk IgG1 Fc, hk hk ( hk' ) Fc pcdna 3.1,, hk Fc,,,,, hk, Ig κ hk 5~10 Z58 11 mg/l, 15~20 mg/l, COS-7 TACI-Fc BCMA-Fc, 2 mg/l [18] ;

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