37 11 215 11 中国预防兽医学报 Chinese Journal of Preventive Veterinary Medicine Vol. 37 No.11 Nov. 215 doi: 1.3969/j.issn.8-589.215.11.12 张瑛杰 1, 尹杰超 1, 李天鹤 2, 周兵 3, 徐鹏飞 1, 荆燕 1,4, 任桂萍 1*, 李德山 1* (1. 153 2. 49 3. 365 4. 153) : 为表达抗鸡传染性法氏囊病病毒 () 抗体 Fab 并检测其中和活性, 本研究将抗 抗体的轻链 (L) 和重链片段 (Fd) 基因分别克隆于 pet-27b (+) 载体中, 并转化于大肠杆菌 Rosetta (DE3) 进行诱导表达将 L 和 Fd 片段包涵体蛋白变性后等量混合于复性液中, 制备 Fab 并对其进行活性鉴定结果显示 L 和 Fd 蛋白相对分子 质量大小分别为 和 28 ku Western blot 和 ELISA 检测结果表明, 获得的抗体 Fab 大小约为 5 ku, 并且与 VP2 蛋白和不同病毒株均具有特异性结合能力体外中和试验结果表明, 获得的 抗体 Fab 具有中和活性, 可以有效阻断 (B87 株 ) 对鸡胚成纤维细胞 (DF1) 的感染本实验获得的 抗体 Fab 有望成为治疗 IBD 的 候选生物制剂, 为研制治疗 IBD 抗体制剂奠定了基础 : 鸡传染性法氏囊病病毒 ;Fab 抗体 ; 共复性 ; 中和活性 ; 治疗性抗体 :S852.65 :A :8-589(215)11-866-5 Expression and test of the neutralization Fab antibody against infectious bursal disease virus ZHANG Ying-jie 1, YIN Jie-chao 1, LI Tian-he 2, ZHOU Bing 3, XU Peng-fei 1, Jing Yan 1,4, REN Gui-ping 1*, LI De-shan 1* (1. Laboratory of Biopharmaceutical, College of Life Science, Northeast Agricultural University, Harbin 153, China; 2. College of Life Science, University of Chinese Academy of Sciences, Beijing 49, China; 3. College of Public Hygiene, Amoy University, Amoy 365, China; 4. Key Laboratory of Agricultural Biological Functional Gene, Northeast Agricultural University, Harbin 153, China) Abstract: To express the neutralizing Fab antibody against infectious bursal disease virus (), the gene of light chain (L) or heavy chain fragment (Fd) against was cloned into the prokaryotic expression plasmid, respectively, and then the recombinant L or Fd was expressed in E.coli Rosetta (DE3), respectively, and purified through sole denaturation and co-renaturation of inclusion body. Western blot results showed that the Fab was approximately 5 ku. ELISA results showed that the Fab exhibited binding ability and specify to VP2 for different strains. The results of neutralization test in vitro showed that the Fab exhibited neutralizing activity to -B87 strainin chicken embryo fibroblast (DF1) cells. The Fab antibody prepared in this study is expected to become a candidate drug for treatment of IBD, which laid the foundation for the treatment of IBD. Key words: infectious bursal disease virus; Fab; corenaturation; neutralizing activity; therapeutic antibody *Corresponding author 215-5-26 (GC13C14) (1991-). * E-mail deshanli@163.com renguiping@126.com
11. Fab 867 (Infectious bursal disease IBD) IBD ().25 mmol/l IPTG 37 4 h [1] SDS-PAGE 1 Table 1 Primers used in the amplification of the target gene Primers Primer sequences (5'-3') P1: Fab-L(F) CCATGGGTGCGCTGACTCAGCCGTCCTCGGTGTC (Nco ) [2] RNA RNA 4 P2: Fab-L(R) GGATCCATTAGCACTCGGACCTCTTCAGGGTCTTC (BamH ) VP2 P3: Fab-Fd(F) CCATGGGTGCCGTGACGTTGGACGAG (Nco ) P4: Fab-Fd(R) GGATCCATTAGAGGACCTGCACCTCTGGGG (BamH ) [3-5] L Fd (8 mol/l ph8.) 1 [6] Fab (L) (Fd) (2 mol/l ph8.) 4 12 h 1/3 5 mmol/l Tris-HCl 48 h L Fd 4 12 r/min 15 min 2 μl [7] PCR Fab L Fd pet-27b(+) L L Fd Fd Fab BSA 1 μl Native-PAGE NC 5 % 37 Fab IBD 2 h VP2 (5 μl/ml) 37 peedual-ires ( ) VP2 DF1 pet-27b (+) E.coli DH5α Rosetta(DE3) Fab 96 Fab (1-65 MB BJ836 NF8 Gt T4 TaqDNA pmd18-t DNA Marker TaKaRa HRP IgG(IgG-HRP) ebioscience Invitrogen ( 1) peedual-ires P1/P2 pet-fd Rosetta (DE3) SDS-PAGE 4 min (1 2) IgG-HRP (1 7 5) Fab 5 μg/ml μg/ml 2 μg/ml 4 μg/ml μl 96 VP2 5 μg/ml (1-65 MB BJ836 NF8 Gt B87 B87 ) PBS 1 VP2 2 3 HRP 4 BSA VP2 5 L 6 Fd 7 3 4 5 % 37 P3/P4 PCR L Fd PCR 2 h VP2 (5 μg/ml) Nco /BamH ( μl) 37 1 h (1 2) pet-27b (+) pet-l IgG-HRP (1 7 5) TMB
868 215 5 min L OD 45nm B87 1 (1-1 ~1-12 ) 96 DF1 8 5 d ~7 d (CPE) Reed-Muench TCID 5 Fab 2 (5 μg/ml~.977 μg/ml) 1 TCID 5 (B87 ) 37 1 h DF1 96 1 1 TCID 5 2 DMEM 3 L 4 Fd 8 37 5 % CO 2 CPE 5 d~7 d Fd ( 2) Rosetta(DE3) A M 1 2 3 4 B M 5 6 7 8 28 ku C M 9 1 11 peedual-ires P1/P2 P3/P4 PCR L Fd pet-27b (+) pet-l pet-fd PCR L Fd 63 bp 7 bp ( 1) M: DL2 DNA Marker; 1: Negative control; 2: L PCR product; 3: Fd PCR product 1 Fig. 1 63 bp M 1 2 3 7 bp pet-l pet-fd PCR PCR product of pet-l and pet-fd plasmid M: Protein Marker; 1, 5: Uninduced bacteria; 2, 6: Induced bacteria; 3, 7: Supernatant of induced bacteria; 4, 8: Precipitation of induced bacteria; 9: L protein; 1: Fd protein; 11: The corenaturation of Fab protein 2 L (A) Fd (B) (C) SDS-PAGE Fig. 2 The expressions of L (A), Fd (B) and purification of the proteins (C) detected by SDS-PAGE Fab ELISA OD 45nm pet-l pet-fd Rosetta(DE3) L Fd Fab BSA Native-PAGE western blot Fab 5 ku VP2 L Fd BSA VP2 ( 3) VP2 Fab PBS (p<.1) SDS-PAGE Fab VP2 Rosetta (DE3) 28 ku Fab 28 ku
11. Fab 869 ( 4) 12 ku 85 ku 5 ku 2 ku M 1 2 3 4 M: Protein Marker; 1: L protein; 2: Fd protein; 3: Fab protein; 4: BSA 2 (B87 ) Fab Table 2 Neutralization tests of the Fab antibody against (B87 strian) Concentration of The percent of CPE (%) Fab (μg/ml) 5 25 125 62.5 31.25 15.625 7.813 3.96 1.953.977 Fab and DMEM L and Fd and 3 Fab western blot Fig. 3 The identification of Fab by western blot A 3. OD45nm 2.5 2. 1.5 1..5. B 2. OD45nm 1.5 1..5. A: Specificity and affinity of Fab to VP2 detected by ELISA; B: Specificity and affinity of Fab to different virus strains detected by ELISA; Note: **p<.1 compared with negative controls 1 2 4 Fig. 4 Fab ELISA Fab detected by ELISA Fab 1 TCID 5 (TCID 5 =1-8.4 /.1 ml) (B87 ) >BJ836 >Gt >MB >NF8 L Fd Fab Fab DF1 (B87 ) DF1 3.96 μg/ml ( 2) 3 4 5 6 7 1 5 μg/ml 2 μg/ml 3 2 μg/ml 4 4 μg/ml 5 PBS 6 Negative 2 7 Negative 3 8 Negative 4 9 Negative 5 1 Negative 6 11 Negative 7 1 2 3 4 5 6 7 8 9 1 1 1 1 2 1 3 8 9 1 1 1-65 strain 2 MB strain 4 BJ836 strain 5 NF8 strain 6 B87 strain 7 PBS 8 Negative 2 9 Negative 3 1 Negative 4 11 Negative 5 12 Negative 6 13 Negative 7 11 [8-1] IBD [8] IBD [11] [12] ( ) [13] PCR Fab L Fd pet-27b(+) Fab Western blot L Fd 5 ku Fab VP2 ELISA Fab VP2 B87 >1-65 3.96 μg/ml Fab
87 215 [14] Fab IBD [1] [2] [3] [4] [5] [6] [7] Hermann M, Md R I, Rudiger R. Research on infectious bursal disease the past, the present and the future [J]. Vet Microbiol, 23, 97(l-2): 153-165.. [J]. virus [J]. Virus Res, 1996, 4(1): 1-15. 1996 16(1) 94-. Parry D A. Coiled-coils in a-helix-containing proteins: analysis of the residue types within the heptad repeat and the use of these data in the prediction of coiled-coils in other proteins [J]. Bio Sci Rep, 1982, 2(12): 117-124. Engelman D M, Steitz T A, Goldman A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins [J]. Ann Rev Biophys Biophys Chem, 1986, 15: 321-353. [11] [12] [13] Sapats S I, Heine H G, Trinidad L, et al. Generation of chicken single chain antibody variable fragments (scfv) that differentiate and neutralize infectious bursal disease virus () [J]. Arch Virol, 23, 148: 497-515. Mine Y, Kovacs-Nolan J. Chicken egg yolk antibodies as therapeutics in enteric infectioudiseasse: a review [J]. J Med Food, 22, 5: 159-169... Gx [J]. [J]. 23 25(2) 14-144. 214 36(3) 227-231.. [14]. Fab [J]. [J]. 25 33(2) 7-73.. Trop-2 Fab [J]. ( ) 212 [8] [9] [1] 32(1) 35-39. Brandt M, Yao Kun, Liu Mei-hong, et al. Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus [J]. J Virol, 21, 75 (24): 11974-11982.. [J]. 1996 61(6) 3-6. Brown M D, Skinner M A. Coding sequences of both genome segments of a European very virulent infectious bursal disease 214 41(3) 318-324. ( )