- Email: [email protected]
Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite
Biochemical and Biophysical Research Communications 393 (2010) 703–707
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite Yuelan Zhao a,1, Said Amer b, Jianyong Wang a, Chengmin Wang c,1, Ying Gao d, Guiying Kang e, Yongzhan Bao d, Hongxuan He c, Jianhua Qin d,* a
College of Chinese Traditional Veterinary Science, Agricultural University of Hebei, Dingzhou 073000, China Department of Zoology, Faculty of Science, Kafr El-Sheikh 33516, Egypt National Research Center for Wildlife Borne Diseases, Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China d College of Animal Science and Technology, Agricultural University of Hebei, Baoding 071001, China e College of Animal Science and Technology, Inner Mongolia University for the Nationalities, Tongliao 028000, China b c
a r t i c l e
i n f o
Article history: Received 4 February 2010 Available online 17 February 2010 Keywords: Eimeria acervulina Merozoite Phage display Single-chain antibody Chicken
a b s t r a c t A single-chain antibody library against Eimeria acervulina merozoites was constructed by phage display approach. Antibody-displaying phage was selected in four panning rounds against cryopreserved E. acervulina merozoites. Five clones were randomly selected from the fourth panning round, and their nucleotide sequences were aligned and compared to mouse germ-line sequences. Soluble antibody was produced in a non-suppressor Escherichia coli strain, purified by protein A affinity chromatography, and characterized by Western-blotting. Immunofluorescence assay showed localization of the produced recombinant antibody fragment on the surface E. acervulina merozoites. These resultant antibody fragments showed high specificity and binding capacity for soluble antigens and intact fixed merozoites which seems promising as diagnostic, therapeutic and/or vaccine tools against coccidiosis. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
Introduction Coccidiosis is a severe disease in chicken and recognized as a serious challenge for the poultry industry [1,2]. It is caused by several species of protozoan parasites belonging to the genus Eimeria [3,4]. Eimeria acervulina (E. acervulina) is one of the most pathogenic Eimeria species in terms of distribution, frequency, and economic losses. In most countries, prophylactic chemotherapy using ionophores and synthetic drugs is still the main method for the control of coccidiosis [5]. However, the long-term use of the preventative anti-coccidial drugs is implicated for the development of drug resistance [6,7] and accumulation of drug residues in livestock products [8,9]. Alternative strategies should be adopted to overcome such drawbacks. Recombinant protein vaccine production is a promising candidate for control of coccidiosis [10]. In this concept, coccidia immune protective antigen protein(s) should be identified, using antibodies with known specificity, to prepare single chain antibodies against chicken coccidiosis [9,11–13]. Yin et al. claimed that it is inevitable to attack coccidia by single-chain antibody–bacteriotoxin conjugates, namely, recombinant immunotoxin [14].
* Corresponding author. Fax: +86 312 7528369. E-mail address: [email protected] (J. Qin). 1 These authors contributed equally to this work.
The initiation of antibody library technology, especially phage display is a promising tool for production of single-chain antibodies [15–17]. In this study, constructed phage antibody library was panned out using E. acervulina merozoites. Single-chain fragment variable antibody (ScFv) was selected and characterized by SDS–PAGE, Western-blotting, ELISA and immunofluorescence assays. Materials and methods Strains and reagents. E. acervulina Baoding strain was provided by Laboratory of Parasitology, College of Animal Science and Technology, Agricultural University of Hebei, China. Escherichia coli strain TG1 was provided by Tiangen Biotech Company Limited (Beijing, China). E. coli HB2151, Helper phage M13KO7, and HRPanti M13IgG were purchased from Pharmacia Corporation (USA). FITC-conjugated goat anti-mouse monoclonal antibody was purchased from Sino-American Biotechnology Co. (Luoyang, China). Preparation of E. acervulina merozoites’ antigen. E. acervulina was propagated by passage through 2-week old broiler chicks. The chicks were inoculated orally with 1.0 104 of sporulated E. acervulina oocysts. Merozoites of the second generation were extracted at 96 h post inoculation and purified [18]. The purified merozoites were used freshly for preparation of soluble antigen or cryopreserved. The soluble antigen of purified merozoites was prepared
0006-291X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.02.063
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Y. Zhao et al. / Biochemical and Biophysical Research Communications 393 (2010) 703–707
using ultrasonic disruption followed by high-speed centrifugation. The protein content of the antigen was estimated spectrophotometrically, adjust to 2.72 mg/ml and stored 20 °C till use. Phage display antibody library construction. The heavy chain (VH) and light chain (VL) antibody genes against E. acervulina merozoites were amplified by RT-PCR using the total RNA, extracted from the spleen cells of BALB/C mice immunized with the soluble antigen of merozoites, as template. The single-chain fragment variable (ScFv) genes of 773 bp with SfiI and NotI restriction sites were amplified with splicing overlap extension-PCR (SOE-PCR) using VL SfiI primer: 50 -CCTTTCTATGCGGCCCAGCCGGCCCAGCCGGCC-30 and VH NotI primer: 50 -TCCGGATACGGCACCGGCGCACCTGCGGCCGC-30 .
The primers were designed by Primer 6.0 software, based on the published sequences (accession No GU235986 to GU235990) of variable regions, VL and VH [19,20]. Purified products were ligated into phagemid pCANTAB5E. Recombinant phagemids were transformed into E. coli TG1 and superinfected by M13KO7 helper phage. Panning and detection. The antibody library was taken through four rounds of panning on E. acervulina cryopreserved merozoites, as described by Abi-Ghanem et al. [21]. In brief, the library was precipitated by addition of 2 ml PEG/NaCl for 30 min on ice bath and collected by centrifugation. Merozoites coated ELISA plates were blocked with 3% Bovine serum albumin (BSA) in PBS and incubated with freshly diluted phage at 37 °C for 1 h. After five
Fig. 1. The nucleotide sequence analysis of the five individual colonies of ScFv. Length of ScFv gene is about 773 bp, encoding 245 amino acids. VL gene in the upstream fragment of about 320 bp in length, while VH gene in the downstream fragment of about 370 bp in length. Length of linker sequence is 51 bp.
Y. Zhao et al. / Biochemical and Biophysical Research Communications 393 (2010) 703–707
times of washing, bound phages were eluted with 0.1 M glycine– HCl, pH 2.2, neutralized with 2 mM Tris–HCl, pH 9.1. Neutralized product was transformed into TG1-Blue E. coli. Transformed bacteria were plated on LB-A medium (50 lg/ml ampicillin) and incubated at 37 °C for 4 h. After four rounds of panning, single colonies of amplified phage were picked up and cultured in LB-AG medium (LB medium containing 2% anhydrous glucose and 50 lg/ml ampicillin) overnight at 30 °C and 250 rpm. After that, the bacteria were cultured in LB-AK medium (LB medium containing 50 lg/ml ampicillin and 50 lg/ml kanamycin) supplemented with 5 108 PFU/mol M13KO7 at 30 °C and 250 rpm for 2 h. The supernatants of the cultures were collected after centrifugation and checked for specificity by ELISA utilizing HRP-anti M13IgG as probe. Sequence analysis. The VH and VL genes amplified by PCR VL SfiI and VH NotI primers and phagemids, from phage-ELISA selection were cloned into pGEM-T Vector and transfected into E. coli TGl. Nucleotide composition of the positive clones was determined using Sanger’s dideoxy sequencing method (Sun-Biotechnology Co., Ltd, Beijing, China). Sequence homology and alignment were performed using DNAstar software. Production of soluble antibody fragments. The positive clones from phage-ELISA selection were transformed into HB2151 competent cells and cultured on SOBAG plates. Single clones selected on SOBAG plates were incubated in LB medium overnight at 37 °C and 250 rpm. After adding 10 ll M13KO7 and 5 ll kanamycin, the bacterial cultures were further incubated at 30 °C and 250 rpm for 3 h. The target fusion proteins were then expressed after induction by isopropyl-b-D-thiogalactopyranoside (IPTG, Sigma) to a final concentration of 1 mmol/l at 30 °C. The expressed products were purified using protein A affinity chromatography [22]. and the concentration of purified antibodies was determined spectrophotometry. Specificity of enriched soluble ScFv was detected was evaluated by ELISA utilizing E. acervulina merozoites antigens coated on 96-well plate as described before. Identity of the expressed ScFv was detected by SDS–PAGE and Western-blotting. Immunofluorescence. Fresh-made merozoites were washed, dried on glass slides, and then fixed in ice cold methanol for 15 min and incubated with the purified anti-E. acervulina ScFv over night at 4 °C. FITC-conjugated goat anti-mouse IgG probe was used at 1:1000 for 40 min, washed and mounted with HardSet Vectashield mounting medium. Processed slides were imaged on a Zeiss xioplan fluorescence microscope for detecting reactivity of ScFv against E. acervulina merozoite antigens [23].
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Fig. 2. SDS–PAGE of E. coli HB2151 expression of Anti-Eimeria acervulian ScFv gene. M: Low MW protein Marker. 1: Empty bacteria control. 2: Non-induced bacteria control. 3–5: Induced products at 2, 4, and 6 h, respectively. 6: Induced product at supernatant. 7–9: Induced products at 8, 10, and 12 h, respectively.
ScFv-3, ScFv-4 and ScFv-5 showed considerable intra-sequence genetic diversity with homology margin of 68.6–80.78% (Fig. 1). Phage antibodies against E. acervulina merozoites After four rounds of panning, single colony of amplified phage was picked up, and the produced ScFvs were tested by ELISA. Results showed that 75% of positive clones produced considerable antibody ELISA titers, 2 h after induction with IPTG (Fig. 2). The expected molecular weight of approximately 32 KDa specific protein band in HB121 strain was confirmed in SDS–PAGE. Western-blotting indicated that ScFv protein showed specific binding activity to merozoite antigens (Fig. 3). The concentration of purified antibodies was 0.89 lg/ml as measured by ultraviolet spectrophotometry. E. acervulina merozoites immunostained by ScFv Reactivity of the four positive ScFv clones against E. acervulina merozoite antigens was determined by immunofluorescence
Results Phage antibody library construction and panning The phages were re-amplified by repeating panning cycles; in this manner specifically binding clones were selected and amplified. Along the four rounds of panning, the phage yield showed a trend of gradual increase as the washing cycles raised from 5 to 20 times, which might indicate that phage display ScFv carrying gene against E. acervulina merozoite surface antigens was condensed. Sequence analysis of ScFv genes against the E. acervulina merozoites The nucleotide sequences of the ScFv genes were obtained and compared with other published ScFvs in the GenBank database. The obtained sequences of 741 bp in length were shown to be consisted of upstream VL sequence of 320 bp, downstream VH sequence of 370 bp and a linker sequence of 51 bp. The generated sequences showed homology with those of VH and VL genes of the ScFv segments characteristic to mice antibody variable genes. Nucleotide sequence analysis of the individual colonies of ScFv-1, ScFv-2,
Fig. 3. Western blot of E. coli HB2151 expression of anti-Eimeria acervulian ScFv. M: Low MW protein Marker. 1–2: Anti-Eimeria acervulian ScFv. 3: M13KO7 negative control.
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Fig. 4. E. acervulina merozoites immunostained by ScFv-A1, A2: Antigen localization on the apical tip and cauda of merozoite; B1, B2: Antigen localization on the on many position of merozoite.
microscopy. Immunofluorescence reactions indicated that epitopes recognized by the ScFv-A1 and ScFv-A2 were localized on the apical tip and cauda of merozoites, while those reacted with ScFv-B1 and ScFv-B2 were randomly localized on merozoites (Fig. 4). Discussion Phage display is a reliable means for the preparation of monoclonal antibodies from both immune and nonimmune sources, without the restraints of the conventional hybridoma approach [24,25]. Furthermore, panning of the constructed library against specific antigen is determinant both on the specificity and phage output [21,26]. Hoogenboom et al. [27] and Da Silva et al. [28] depicted similar conclusion. Non-suppressor E. coli HB2151 strain was infected by phagemid pCANTAB5E DNA and subsequently soluble proteins were secreted directly into the culture supernatant [29]. Herein, this cloning system was used to express functional fragments of antibodies by using VL-Linker-VH connection. The results indicated that the high-level expression of ScFv was achieved which could be detected at 2 h after induction, and sustained up to 16 h. Production of 32 KDa specific ScFvs was confirmed by SDS– PAGE and Western-blotting, testes. Moreover, high specific binding capacities were confirmed against soluble E. acervulina merozites’ antigens using ELISA. The immunogenic epitopes might be dominant on the surface of merozoites as revealed by immunofluorescence localization. Sequence results of the selected clones showed considerable intra-sequence genetic diversity with homology margin of 68.6–80.78%. These results fully agree with those reported by Wieland et al. [30] and Abi-Ghanem et al. [21] using E. tenella-sporozoites system. Diversification at VL–VH repertoire
may be attributed to possible donor pseudogenes and/or possible somatic hypermutation events [21,31]. Taken together, phage display might be a valid approach for production of monoclonal antibodies/antibody fragments with different specificities for various developmental stages and the possibility of application with other coccidia models. Further investigations are needed to ascertain ScFv anti-coccidial protein structure and function as well as to identify the possibility of its usage as a diagnostic, therapeutic and/or vaccine candidate against coccidiosis [28]. Acknowledgments This research was supported by grants from the National Natural Science Foundation of China (Grant number: 30571394). The authors thank Prof. Wang Ming (College of Veterinary Medicine, China Agricultural University, Beijing, China) for technical assistance. References [1] P.C. Augustine, Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria, Int. J. Parasitol. 31 (1) (2001) 1–8. [2] R.A. Dalloul, H.S. Lillehoj, Poultry coccidiosis: recent advancements in control measures and vaccine development, Expert Rev. Vaccines 5 (1) (2006) 143– 163. [3] V. McDonald, M.W. Shirley, Past and future: vaccination against Eimeria, Parasitology 136 (12) (2009) 1477–1489. [4] R.B. Williams, A compartmentalised model for the estimation of the cost of coccidiosis to the world’s chicken production industry, Int. J. Parasitol. 29 (8) (1999) 1209–1229. [5] H.D. Chapman, A landmark contribution to poultry science – prophylactic control of coccidiosis in poultry, Poult. Sci. 88 (4) (2009) 813–815.
Y. Zhao et al. / Biochemical and Biophysical Research Communications 393 (2010) 703–707 [6] H.D. Chapman, Biochemical, genetic and applied aspects of drug resistance in Eimeria parasites of the fowl, Avian Pathol. 26 (2) (1997) 221–244. [7] A. Haberkorn, Chemotherapy of human, animal coccidioses: state and perspectives, Parasitol. Res. 82 (3) (1996) 193–199. [8] H.D. Chapman, Anticoccidial drugs and their effects upon the development of immunity to Eimeria infections in poultry, Avian Pathol. 28 (6) (1999) 521– 535. [9] H.S. Lillehoj, E.P. Lillehoj, Coccidiosis: a review of acquired intestinal immunity and vaccination strategies, Avian Dis. 44 (2) (2000) 408–425. [10] X. Song, L. Xu, R. Yan, X. Huang, M.A. Shah, X. Li, The optimal immunization procedure of DNA vaccine pcDNA-TA4-IL-2 of Eimeria tenella and its crossimmunity to Eimeria necatrix and Eimeria acervulina, Vet. Parasitol. 159 (1) (2009) 30–36. [11] M.D. Castle, M.C. Jenkins, H.D. Danforth, H.S. Lillehoj, Characterization of a recombinant Eimeria acervulina antigen expressed in sporozoite and merozoite developmental stages, J. Parasitol. 77 (3) (1991) 384–390. [12] D.P. Blake, M.W. Shirley, A.L. Smith, Genetic identification of antigens protective against coccidia, Parasite Immunol. 28 (7) (2006) 305–314. [13] A.N. Vermeulen, Progress in recombinant vaccine development against coccidiosis a review and prospects into the next millennium, Int. J. Parasitol. 28 (7) (1998) 1121–1130. [14] J.G. Yin, X.C. Zhang, J.H. Li, P. Zhu, Z.S. Liu, Construction of anti-Cryptosporidium parvum sporozoite immunotoxin ScFv-PE40 and expression in E. coli, Acta Parasi. Med Entomol Sinica (in Chinese) 12 (3) (2005) 139–142.. [15] G. Winter, A.D. Griffiths, R.E. Hawkins, H.R. Hoogenboom, Making antibodies by phage display technology, Annu. Rev. Immunol. 12 (1994) 433–455. [16] T. Bratkovic, Progress in phage display evolution of the technique and its applications, Cell. Mol. Life Sci. (2009), doi:10.1007/s00018-009-0192-2. [17] K. Onodera, Molecular biology and biotechnology of bacteriophage, Adv. Biochem. Eng. Biotechnol. (2009), doi:10.1007/10_2008_46. [18] M.A. Fernando, Fine structure of the schizonts and merozoites of Eimeria acervulina in the chicken, J. Parasitol. 60 (1) (1974) 149–159. [19] M. He, N. Cooley, A. Jackson, M.J. Taussig, Production of human singlechain antibodies by ribosome display, Methods Mol. Biol. 248 (2004) 177– 189. [20] S. Imai, Y. Mukai, K. Nagano, H. Shibata, T. Sugita, Y. Abe, T. Nomura, Y. Tsutsumi, H. Kamada, S. Nakagawa, S. Tsunoda, Quality enhancement of the non-immune phage scFv library to isolate effective antibodies, Biol. Pharm. Bull. 29 (7) (2006) 1325–1330.
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[21] D. Abi-Ghanem, S.D. Waghela, D.J. Caldwell, H.D. Danforth, L.R. Berghman, Phage display selection and characterization of single-chain recombinant antibodies against Eimeria tenella sporozoites, Vet. Immunol. Immunopathol. 121 (1–2) (2008) 58–67. [22] H.L. Zhou, M.M. Lü, N. Wang, Z. Liu, J.G. Zhang, Purification of monoclonal antibodies using protein A affinity chromatography, Biotechnol. Bull. 5 (2005) 72–74. [23] Y.J. Chen, X.Q. Feng, S.Q. Zhong, Z.W. Pan, Expression of protein kinase CA in preimplantation mouse embryos by confocal laser microscopy, J. Harbin Med. University (in Chinese) 41 (3) (2007) 196–198. [24] J. Andris-Widhopf, C. Rader, P. Steinberger, R. Fuller, C.F. Barbas III., Methods for the generation of chicken monoclonal antibody fragments by phage display, J. Immunol. Methods 242 (1–2) (2000) 159–181. [25] S. Deckers, I. Braren, K. Greunke, N. Meyer, D. Rühl, R. Bredehorst, E. Spillner, Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries, Biotechnol. Appl. Biochem. 52 (Pt 1) (2009) 79–87. [26] M.E. Kabir, S. Krishnaswamy, M. Miyamoto, Y. Furuichi, T. Komiyama, An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody, BMC Biotechnol. 9 (1) (2009) 99, doi:10.1186/14726750-9-99. [27] H.R. Hoogenboom, A.D. Griffiths, K.S. Johnson, D.J. Chiswell, P. Hudson, G. Winter, Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody(Fab) heavy and light chains, Nucleic Acids Res. 19 (15) (1991) 4133–4137. [28] A.J. Da Silva, U. Kawazoe, F.F. Freitas, M.S. Gatti, H. Dolder, R.I. Schumacher, M.A. Juliano, M.J. da Silva, A. Leite, Avian anticoccidial activity of a novel membrane-interactive peptide selected from phage display libraries, Mol. Biochem. Parasitol. 120 (1) (2002) 53–60. [29] C.F. Barbas III., A.S. Kang, R.A. Lenner, S.J. Benkovic, Assembly of combinatorial antibody libraries on phage surface: the gene III site, Proc. Natl. Acad. Sci. USA 88 (18) (1991) 7978–7982. [30] W.H. Wieland, D. Orzáez, A. Lammers, H.K. Parmentier, A. Schots, Display and selection of chicken IgA Fab fragments, Vet. Immunol. Immunopathol. 110 (1– 2) (2006) 129–140. [31] K.J. Park, D.W. Park, C.H. Kim, B.K. Han, T.S. Park, J.Y. Han, H.S. Lillehoj, J.K. Kim, Development and characterization of a recombinant chicken single-chain Fv antibody detecting Eimeria acervulina sporozoite antigen, Biotechnol. Lett. 27 (5) (2005) 289–295.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite Yuelan Zhao a,1, Said Amer b, Jianyong Wang a, Chengmin Wang c,1, Ying Gao d, Guiying Kang e, Yongzhan Bao d, Hongxuan He c, Jianhua Qin d,* a
College of Chinese Traditional Veterinary Science, Agricultural University of Hebei, Dingzhou 073000, China Department of Zoology, Faculty of Science, Kafr El-Sheikh 33516, Egypt National Research Center for Wildlife Borne Diseases, Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China d College of Animal Science and Technology, Agricultural University of Hebei, Baoding 071001, China e College of Animal Science and Technology, Inner Mongolia University for the Nationalities, Tongliao 028000, China b c
a r t i c l e
i n f o
Article history: Received 4 February 2010 Available online 17 February 2010 Keywords: Eimeria acervulina Merozoite Phage display Single-chain antibody Chicken
a b s t r a c t A single-chain antibody library against Eimeria acervulina merozoites was constructed by phage display approach. Antibody-displaying phage was selected in four panning rounds against cryopreserved E. acervulina merozoites. Five clones were randomly selected from the fourth panning round, and their nucleotide sequences were aligned and compared to mouse germ-line sequences. Soluble antibody was produced in a non-suppressor Escherichia coli strain, purified by protein A affinity chromatography, and characterized by Western-blotting. Immunofluorescence assay showed localization of the produced recombinant antibody fragment on the surface E. acervulina merozoites. These resultant antibody fragments showed high specificity and binding capacity for soluble antigens and intact fixed merozoites which seems promising as diagnostic, therapeutic and/or vaccine tools against coccidiosis. Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved.
Introduction Coccidiosis is a severe disease in chicken and recognized as a serious challenge for the poultry industry [1,2]. It is caused by several species of protozoan parasites belonging to the genus Eimeria [3,4]. Eimeria acervulina (E. acervulina) is one of the most pathogenic Eimeria species in terms of distribution, frequency, and economic losses. In most countries, prophylactic chemotherapy using ionophores and synthetic drugs is still the main method for the control of coccidiosis [5]. However, the long-term use of the preventative anti-coccidial drugs is implicated for the development of drug resistance [6,7] and accumulation of drug residues in livestock products [8,9]. Alternative strategies should be adopted to overcome such drawbacks. Recombinant protein vaccine production is a promising candidate for control of coccidiosis [10]. In this concept, coccidia immune protective antigen protein(s) should be identified, using antibodies with known specificity, to prepare single chain antibodies against chicken coccidiosis [9,11–13]. Yin et al. claimed that it is inevitable to attack coccidia by single-chain antibody–bacteriotoxin conjugates, namely, recombinant immunotoxin [14].
* Corresponding author. Fax: +86 312 7528369. E-mail address: [email protected] (J. Qin). 1 These authors contributed equally to this work.
The initiation of antibody library technology, especially phage display is a promising tool for production of single-chain antibodies [15–17]. In this study, constructed phage antibody library was panned out using E. acervulina merozoites. Single-chain fragment variable antibody (ScFv) was selected and characterized by SDS–PAGE, Western-blotting, ELISA and immunofluorescence assays. Materials and methods Strains and reagents. E. acervulina Baoding strain was provided by Laboratory of Parasitology, College of Animal Science and Technology, Agricultural University of Hebei, China. Escherichia coli strain TG1 was provided by Tiangen Biotech Company Limited (Beijing, China). E. coli HB2151, Helper phage M13KO7, and HRPanti M13IgG were purchased from Pharmacia Corporation (USA). FITC-conjugated goat anti-mouse monoclonal antibody was purchased from Sino-American Biotechnology Co. (Luoyang, China). Preparation of E. acervulina merozoites’ antigen. E. acervulina was propagated by passage through 2-week old broiler chicks. The chicks were inoculated orally with 1.0 104 of sporulated E. acervulina oocysts. Merozoites of the second generation were extracted at 96 h post inoculation and purified [18]. The purified merozoites were used freshly for preparation of soluble antigen or cryopreserved. The soluble antigen of purified merozoites was prepared
0006-291X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.02.063
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Y. Zhao et al. / Biochemical and Biophysical Research Communications 393 (2010) 703–707
using ultrasonic disruption followed by high-speed centrifugation. The protein content of the antigen was estimated spectrophotometrically, adjust to 2.72 mg/ml and stored 20 °C till use. Phage display antibody library construction. The heavy chain (VH) and light chain (VL) antibody genes against E. acervulina merozoites were amplified by RT-PCR using the total RNA, extracted from the spleen cells of BALB/C mice immunized with the soluble antigen of merozoites, as template. The single-chain fragment variable (ScFv) genes of 773 bp with SfiI and NotI restriction sites were amplified with splicing overlap extension-PCR (SOE-PCR) using VL SfiI primer: 50 -CCTTTCTATGCGGCCCAGCCGGCCCAGCCGGCC-30 and VH NotI primer: 50 -TCCGGATACGGCACCGGCGCACCTGCGGCCGC-30 .
The primers were designed by Primer 6.0 software, based on the published sequences (accession No GU235986 to GU235990) of variable regions, VL and VH [19,20]. Purified products were ligated into phagemid pCANTAB5E. Recombinant phagemids were transformed into E. coli TG1 and superinfected by M13KO7 helper phage. Panning and detection. The antibody library was taken through four rounds of panning on E. acervulina cryopreserved merozoites, as described by Abi-Ghanem et al. [21]. In brief, the library was precipitated by addition of 2 ml PEG/NaCl for 30 min on ice bath and collected by centrifugation. Merozoites coated ELISA plates were blocked with 3% Bovine serum albumin (BSA) in PBS and incubated with freshly diluted phage at 37 °C for 1 h. After five
Fig. 1. The nucleotide sequence analysis of the five individual colonies of ScFv. Length of ScFv gene is about 773 bp, encoding 245 amino acids. VL gene in the upstream fragment of about 320 bp in length, while VH gene in the downstream fragment of about 370 bp in length. Length of linker sequence is 51 bp.
Y. Zhao et al. / Biochemical and Biophysical Research Communications 393 (2010) 703–707
times of washing, bound phages were eluted with 0.1 M glycine– HCl, pH 2.2, neutralized with 2 mM Tris–HCl, pH 9.1. Neutralized product was transformed into TG1-Blue E. coli. Transformed bacteria were plated on LB-A medium (50 lg/ml ampicillin) and incubated at 37 °C for 4 h. After four rounds of panning, single colonies of amplified phage were picked up and cultured in LB-AG medium (LB medium containing 2% anhydrous glucose and 50 lg/ml ampicillin) overnight at 30 °C and 250 rpm. After that, the bacteria were cultured in LB-AK medium (LB medium containing 50 lg/ml ampicillin and 50 lg/ml kanamycin) supplemented with 5 108 PFU/mol M13KO7 at 30 °C and 250 rpm for 2 h. The supernatants of the cultures were collected after centrifugation and checked for specificity by ELISA utilizing HRP-anti M13IgG as probe. Sequence analysis. The VH and VL genes amplified by PCR VL SfiI and VH NotI primers and phagemids, from phage-ELISA selection were cloned into pGEM-T Vector and transfected into E. coli TGl. Nucleotide composition of the positive clones was determined using Sanger’s dideoxy sequencing method (Sun-Biotechnology Co., Ltd, Beijing, China). Sequence homology and alignment were performed using DNAstar software. Production of soluble antibody fragments. The positive clones from phage-ELISA selection were transformed into HB2151 competent cells and cultured on SOBAG plates. Single clones selected on SOBAG plates were incubated in LB medium overnight at 37 °C and 250 rpm. After adding 10 ll M13KO7 and 5 ll kanamycin, the bacterial cultures were further incubated at 30 °C and 250 rpm for 3 h. The target fusion proteins were then expressed after induction by isopropyl-b-D-thiogalactopyranoside (IPTG, Sigma) to a final concentration of 1 mmol/l at 30 °C. The expressed products were purified using protein A affinity chromatography [22]. and the concentration of purified antibodies was determined spectrophotometry. Specificity of enriched soluble ScFv was detected was evaluated by ELISA utilizing E. acervulina merozoites antigens coated on 96-well plate as described before. Identity of the expressed ScFv was detected by SDS–PAGE and Western-blotting. Immunofluorescence. Fresh-made merozoites were washed, dried on glass slides, and then fixed in ice cold methanol for 15 min and incubated with the purified anti-E. acervulina ScFv over night at 4 °C. FITC-conjugated goat anti-mouse IgG probe was used at 1:1000 for 40 min, washed and mounted with HardSet Vectashield mounting medium. Processed slides were imaged on a Zeiss xioplan fluorescence microscope for detecting reactivity of ScFv against E. acervulina merozoite antigens [23].
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Fig. 2. SDS–PAGE of E. coli HB2151 expression of Anti-Eimeria acervulian ScFv gene. M: Low MW protein Marker. 1: Empty bacteria control. 2: Non-induced bacteria control. 3–5: Induced products at 2, 4, and 6 h, respectively. 6: Induced product at supernatant. 7–9: Induced products at 8, 10, and 12 h, respectively.
ScFv-3, ScFv-4 and ScFv-5 showed considerable intra-sequence genetic diversity with homology margin of 68.6–80.78% (Fig. 1). Phage antibodies against E. acervulina merozoites After four rounds of panning, single colony of amplified phage was picked up, and the produced ScFvs were tested by ELISA. Results showed that 75% of positive clones produced considerable antibody ELISA titers, 2 h after induction with IPTG (Fig. 2). The expected molecular weight of approximately 32 KDa specific protein band in HB121 strain was confirmed in SDS–PAGE. Western-blotting indicated that ScFv protein showed specific binding activity to merozoite antigens (Fig. 3). The concentration of purified antibodies was 0.89 lg/ml as measured by ultraviolet spectrophotometry. E. acervulina merozoites immunostained by ScFv Reactivity of the four positive ScFv clones against E. acervulina merozoite antigens was determined by immunofluorescence
Results Phage antibody library construction and panning The phages were re-amplified by repeating panning cycles; in this manner specifically binding clones were selected and amplified. Along the four rounds of panning, the phage yield showed a trend of gradual increase as the washing cycles raised from 5 to 20 times, which might indicate that phage display ScFv carrying gene against E. acervulina merozoite surface antigens was condensed. Sequence analysis of ScFv genes against the E. acervulina merozoites The nucleotide sequences of the ScFv genes were obtained and compared with other published ScFvs in the GenBank database. The obtained sequences of 741 bp in length were shown to be consisted of upstream VL sequence of 320 bp, downstream VH sequence of 370 bp and a linker sequence of 51 bp. The generated sequences showed homology with those of VH and VL genes of the ScFv segments characteristic to mice antibody variable genes. Nucleotide sequence analysis of the individual colonies of ScFv-1, ScFv-2,
Fig. 3. Western blot of E. coli HB2151 expression of anti-Eimeria acervulian ScFv. M: Low MW protein Marker. 1–2: Anti-Eimeria acervulian ScFv. 3: M13KO7 negative control.
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Fig. 4. E. acervulina merozoites immunostained by ScFv-A1, A2: Antigen localization on the apical tip and cauda of merozoite; B1, B2: Antigen localization on the on many position of merozoite.
microscopy. Immunofluorescence reactions indicated that epitopes recognized by the ScFv-A1 and ScFv-A2 were localized on the apical tip and cauda of merozoites, while those reacted with ScFv-B1 and ScFv-B2 were randomly localized on merozoites (Fig. 4). Discussion Phage display is a reliable means for the preparation of monoclonal antibodies from both immune and nonimmune sources, without the restraints of the conventional hybridoma approach [24,25]. Furthermore, panning of the constructed library against specific antigen is determinant both on the specificity and phage output [21,26]. Hoogenboom et al. [27] and Da Silva et al. [28] depicted similar conclusion. Non-suppressor E. coli HB2151 strain was infected by phagemid pCANTAB5E DNA and subsequently soluble proteins were secreted directly into the culture supernatant [29]. Herein, this cloning system was used to express functional fragments of antibodies by using VL-Linker-VH connection. The results indicated that the high-level expression of ScFv was achieved which could be detected at 2 h after induction, and sustained up to 16 h. Production of 32 KDa specific ScFvs was confirmed by SDS– PAGE and Western-blotting, testes. Moreover, high specific binding capacities were confirmed against soluble E. acervulina merozites’ antigens using ELISA. The immunogenic epitopes might be dominant on the surface of merozoites as revealed by immunofluorescence localization. Sequence results of the selected clones showed considerable intra-sequence genetic diversity with homology margin of 68.6–80.78%. These results fully agree with those reported by Wieland et al. [30] and Abi-Ghanem et al. [21] using E. tenella-sporozoites system. Diversification at VL–VH repertoire
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