This project was funded by the Modern Agricultural Industry Technology System Foundation of Shandong Province (SDAIT-09-011-06), the Chinese Medicine Antiviral Collaborative Innovation Center of Shandong Province Colleges (XTCX2014B0108), the Science and Technology Development Plan of Shandong Province (2012GNC11020), and the Natural Science Foundation of Shandong Province (ZR2015CQ019 and ZR2014CM044).
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REFERENCES
Amigorena, S., and Bonnerot, C. (1999). Fc receptors for IgG and antigen presentation on MHC class I and class II molecules.Semin. Immunol.11, 385–390. doi: 10.1006/smim.1999.0196
Brumeanu, T., Swiggard, W., Steinman, R., Bona, C., and Zaghouani, H. (1993).
Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus. J. Exp. Med. 178, 1795–1799. doi:
10.1084/jem.178.5.1795
Cereghino, J. L., and Cregg, J. M. (2000). Heterologous protein expression in the methylotrophic yeastPichia pastoris.Fems. Microbiol. Rev.24, 45–66. doi:
10.1111/j.1574-6976.2000.tb00532.x
Cook, J., and Barber, B. H. (1995). Recombinant antibodies containing an engineered B-cell epitope capable of eliciting conformation-specific antibody responses.Vaccine13, 1770–1778. doi: 10.1016/0264-410X(95)00140-V Cui, G., Zhong, S., Yang, S., Zuo, X., Liang, M., Sun, J., et al. (2013).
Effects of TaishanPinus massonianapollen polysaccharide on the subunit vaccine ofProteus mirabilisin birds.Int. J. Biol. Macromol.56, 94–98. doi:
10.1016/j.ijbiomac.2013.02.006
Denac, H., Moser, C., Tratschin, J. D., and Hofmann, M. A. (1997). An indirect ELISA for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen.
J. Virol. Methods.65, 169–181. doi: 10.1016/S0166-0934(97)02186-1
Ding, A. H., Nathan, C. F., and Stuehr, D. J. (1988). Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production.J. Immunol.141, 2407–2412.
Gentry-Weeks, C. R., Hultsch, A.-L., Kelly, S. M., Keith, J. M., and Curtiss, R.
(1992). Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.J. Bacteriol.174, 7729–7742.
Harrington, A. T., Castellanos, J. A., Ziedalski, T. M., Clarridge, J. E., and Cookson, B. T. (2009). Isolation of Bordetella aviumand novelBordetella strain from patients with respiratory disease.Emerg. Infect. Dis15, 72–74. doi:
10.3201/eid1501.071677
Henneke, P., and Golenbock, D. T. (2004). Phagocytosis, innate immunity, and host–pathogen specificity.J. Exp. Med.199, 1–4. doi: 10.1084/jem.20031256 Hu, X., Zhu, R., Liu, H., Bi, J., and Li, X. (2007). Study on extraction
and antigenicity of the outer-membrane-protein of Bordetella avium.Acta Microbiol. Sin.47, 714–717.
Iorio, A., Krishnan, S., Myrén, K., Lethagen, S., McCormick, N., and Karner, P.
(2015). Factor consumption for prophylaxis and treatment of bleeding:
recombinant factor Ix Fc fusion protein compared with conventional recombinant factor Ix. Value Health 18:A660. doi: 10.1016/j.jval.2015.
09.2396
Jalava, K., Hensel, A., Szostak, M., Resch, S., and Lubitz, W. (2002). Bacterial ghosts as vaccine candidates for veterinary applications.J. Control. Release85, 17–25.
doi: 10.1016/S0168-3659(02)00267-5
Jefferis, R. (2009). Glycosylation as a strategy to improve antibody-based therapeutics.Nat. Rev. Drug Discov.8, 226–234. doi: 10.1038/nrd2804 Jiang, J., Wu, C., Gao, H., Song, J., and Li, H. (2010). Effects of astragalus
polysaccharides on immunologic function of erythrocyte in chickens infected with infectious bursa disease virus. Vaccine 28, 5614–5616. doi:
10.1016/j.vaccine.2010.06.025
Konduru, K., Bradfute, S. B., Jacques, J., Manangeeswaran, M., Nakamura, S., Morshed, S., et al. (2011). Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice.Vaccine29, 2968–2977.
doi: 10.1016/j.vaccine.2011.01.113
Liu, L., Yu, C., Wang, C., Shao, M., Yan, Z., Jiang, X., et al. (2016).
Immuno-enhancement of TaishanPinus massonianapollen polysaccharides on recombinantBordetella aviumompA expressed inPichia pastoris.Microb.
Pathog.95, 54–61. doi: 10.1016/j.micpath.2016.03.002
Lowry, S. F. (1993). Cytokine mediators of immunity and inflammation.Arch. Surg.
128, 1235–1241. doi: 10.1001/archsurg.1993.01420230063010
Lu, L., Palaniyandi, S., Zeng, R., Bai, Y., Liu, X., Wang, Y., et al. (2011).
A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection.J. Virol.85, 10542–10553.
doi: 10.1128/JVI.05441-11
Ma, X., Guo, Z., Wang, D., Hu, Y., and Shen, Z. (2010). Effects of sulfated polysaccharides and their prescriptions on immune response of ND vaccine in chicken.Carbohydr. Polym.82, 9–13. doi: 10.1016/j.carbpol.2010.04.013 Mahapatra, S. K., Das, S., Bhattacharjee, S., Gautam, N., Majumdar, S., and
Roy, S. (2009). In vitro nicotine-induced oxidative stress in mice peritoneal macrophages: a dose-dependent approach.Mech. Method.19, 100–108. doi:
10.1080/15376510802255184
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays.J. Immunol. Methods65, 55–63. doi: 10.1016/0022-1759(83)90303-4
Mwanza, M., Kametler, L., Bonai, A., Rajli, V., Kovacs, M., and Dutton, M. F.
(2009). The cytotoxic effect of fumonisin B1 and ochratoxin a on human and pig lymphocytes using the Methyl Thiazol Tetrazolium (MTT) assay.Mycotoxin Res.25, 233–238. doi: 10.1007/s12550-009-0033-z
Neild, A., Murata, T., and Roy, C. R. (2005). Processing and major histocompatibility complex class II presentation of Legionella pneumophila antigens by infected macrophages.Infect. Immun.73, 2336–2343. doi: 10.1128/
IAI.73.4.2336-2343.2005
Nimmerjahn, F., and Ravetch, J. V. (2008). Fcγreceptors as regulators of immune responses.Nat. Rev. Immunol.8, 34–47. doi: 10.1038/nri2206
Peng, W., Si, W., Yin, L., Liu, H., Yu, S., Liu, S., et al. (2011). Salmonella enteritidis ghost vaccine induces effective protection against lethal challenge in specific-pathogen-free chicks. Immunobiology 216, 558–565.
doi: 10.1016/j.imbio.2010.10.001
Qin, M., Tang, X., Yin, G., Liu, X., Suo, J., and Geru, T. (2016). Chicken igy fc expressed by eimeria mitis enhances the immunogenicity of e. mitis.Parasit.
Vectors9, 1–7. doi: 10.1186/s13071-016-1451-3
Rath, T., Baker, K., Dumont, J. A., Peters, R. T., Jiang, H., Qiao, S.-W., et al.
(2015). Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics. Crit. Rev. Biotechnol. 35, 235–254. doi:
10.3109/07388551.2013.834293
Roopenian, D. C., and Akilesh, S. (2007). FcRn: the neonatal Fc receptor comes of age.Nat. Rev. Immunol.7, 715–725. doi: 10.1038/nri2155
Saif, Y. M., Moorhead, P., and Whitmoyer, R. (1981). Scanning electron microscopy of tracheas from turkey poults infected withAlcaligenes faecalis.
Avian Dis.25, 730–735. doi: 10.2307/1590004
Schepetkin, I. A., and Quinn, M. T. (2006). Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Int. Immunopharmacol. 6, 317–333. doi: 10.1016/j.intimp.2005.10.005
Sun, H., Zhang, J., Chen, F., Chen, X., Zhou, Z., and Wang, H. (2015). Activation of RAW264. 7 macrophages by the polysaccharide from the roots ofActinidia erianthaand its molecular mechanisms.Carbohydr. Polym.121, 388–402. doi:
10.1016/j.carbpol.2014.12.023
Sutterwala, F. S., Noel, G. J., Clynes, R., and Mosser, D. M. (1997). Selective suppression of interleukin-12 induction after macrophage receptor ligation.
J. Exp. Med.185, 1977–1985. doi: 10.1084/jem.185.11.1977
Tayra, J. T., Kameda, M., Yasuhara, T., Agari, T., Kadota, T., Wang, F., et al. (2013).
The neuroprotective and neurorescue effects of carbamylated erythropoietin Fc fusion protein (CEPO-Fc) in a rat model of Parkinson’s disease.Brain Res.1502, 55–70. doi: 10.1016/j.brainres.2013.01.042
Temple, L. M., Miyamoto, D. M., Mehta, M., Capitini, C. M., Von Stetina, S., Barnes, H. J., et al. (2010). Identification and characterization of twoBordetella avium gene products required for hemagglutination. Infect. Immun. 78, 2370–2376. doi: 10.1128/IAI.00140-10
Torti, C., Prosperi, M., Motta, D., Digiambenedetto, S., Maggiolo, F., Paraninfo, G., et al. (2012). Factors influencing the normalization of CD4+T-cell count, percentage and CD4+/CD8+T-cell ratio in HIV-infected patients on long- term suppressive antiretroviral therapy.Clin. Microbiol. Infec.18, 449–458. doi:
10.1111/j.1469-0691.2011.03650.x
Wei, K., Sun, Z., Yan, Z., Tan, Y., Wang, H., Zhu, X., et al. (2011).
Effects of Taishan Pinus massoniana pollen polysaccharide on immune response of rabbit haemorrhagic disease tissue inactivated vaccine and on production performance of Rex rabbits. Vaccine 29, 2530–2536. doi:
10.1016/j.vaccine.2011.01.068
Yang, S., Wei, K., Jia, F., Zhao, X., Cui, G., Guo, F., et al. (2015).
Characterization and biological activity of TaishanPinus massonianapollen polysaccharide in vitro.PLoS ONE10:e0115638. doi: 10.1371/journal.pone.01 15638
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Zaharatos, G. J., Yu, J., Pace, C., Song, Y., Vasan, S., Ho, D. D., et al. (2011). HIV-1 and influenza antigens synthetically linked to IgG2a Fc elicit superior humoral responses compared to unmodified antigens in mice.Vaccine30, 42–50. doi:
10.1016/j.vaccine.2011.10.05
Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Copyright © 2016 Dong, Zhang, Huang, Zhou, Hu, Lian, Zhu, Ma, Yang, Wei and Zhu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Frontiers in Microbiology | www.frontiersin.org 295 November 2016 | Volume 7 | Article 1708
ORIGINAL RESEARCH published: 29 September 2016 doi: 10.3389/fmicb.2016.01537
Frontiers in Microbiology | www.frontiersin.org September 2016 | Volume 7 | Article 1537
Edited by:
Julio Villena, Reference Centre for Lactobacilli (CERELA-CONICET), Argentina
Reviewed by:
Hridayesh Prakash, University of Hyderabad, India M. Victoria Delpino, Institute of Immunology, Genetics, and Metabolism (CONICET), Argentina
*Correspondence:
Sabina Górska [email protected]
Specialty section:
This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology
Received:05 May 2016 Accepted:14 September 2016 Published:29 September 2016
Citation:
Górska S, Dylus E, Rudawska A, Brzozowska E, Srutkova D, Schwarzer M, Razim A, Kozakova H and Gamian A (2016) Immunoreactive Proteins of Bifidobacterium longum ssp. longum CCM 7952 and Bifidobacterium longum ssp. longum CCDM 372 Identified by Gnotobiotic Mono-Colonized Mice Sera, Immune Rabbit Sera and Non-immune Human Sera. Front. Microbiol. 7:1537.
doi: 10.3389/fmicb.2016.01537
Immunoreactive Proteins of
Bifidobacterium longum ssp. longum CCM 7952 and Bifidobacterium
longum ssp. longum CCDM 372 Identified by Gnotobiotic
Mono-Colonized Mice Sera, Immune Rabbit Sera and Non-immune Human Sera
Sabina Górska1*, Ewa Dylus1, Angelika Rudawska1, Ewa Brzozowska1, Dagmar Srutkova2, Martin Schwarzer2, Agnieszka Razim1, Hana Kozakova2and Andrzej Gamian1
1Department of Medical Microbiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, Wroclaw, Poland,2Laboratory of Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic v. v. i., Novy Hradek, Czech Republic
The Bifidobacteria show great diversity in the cell surface architecture which may influence the physicochemical properties of the bacterial cell and strain specific properties. The immunomodulatory role of bifidobacteria has been extensively studied, however studies on the immunoreactivity of their protein molecules are very limited.
Here, we compared six different methods of protein isolation and purification and we report identification of immunogenic and immunoreactive protein of two human Bifidobacterium longum ssp. longum strains. We evaluated potential immunoreactive properties of proteins employing polyclonal sera obtained from germ free mouse, rabbit and human. The protein yield was isolation method-dependent and the reactivity of proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and have them sequenced. Among the immunoreactive proteins we identified enolase, aspartokinase, pyruvate kinase, DnaK (B. longum ssp. longum CCM 7952) and sugar ABC transporter ATP-binding protein, phosphoglycerate kinase, peptidoglycan synthethase penicillin-binding protein 3, transaldolase, ribosomal proteins and glyceraldehyde 3-phosphate dehydrogenase (B. longumssp.longumCCDM 372).
Keywords:Bifidobacterium, probiotics, moonlighting proteins, immunoreactivity, surface proteins
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Górska et al. Immunoreactive Proteins ofBifidobacterium
INTRODUCTION
Bifidobacteria are nonpathogenic, anaerobic, non-motile, non- sporulating, non-gas producing, branched rod-shaped Gram- positive bacteria highly represented in the normal microbiota of the human and animals gastrointestinal tract. They were first isolated from feces of a breast-fed infant by Tissier in 1899, and then named Bacillus bifidus (Tisseir, 1900). The Bifidobacterium genus comprises 48 species, exhibits a wide spectrum of metabolic properties and possess genomes of a high G+C content (60%) (Ventura et al., 2007; Endo et al., 2012;
Killer et al., 2009, 2011). They are widely used in food and pharmaceutical industry as a synbiotic or probiotics due to their health-promoting effects on the human host (Gaggia et al., 2011).
Certain Bifidobacteria exert a range of health benefits, including the regulation of intestinal microbial homeostasis, the inhibition of pathogens and harmful bacteria that colonize and/or infect the gut mucosa, the modulation of local and systemic immune responses, the repression of procarcinogenic enzymatic activities within the microbiota, the production of vitamins, and the bioconversion of a number of dietary compounds into bioactive molecules (Singh et al., 1997; Picard et al., 2005). It has been already accepted that probiotic effects ofBifidobacteriumstrain is strictly strain dependent and should not be assigned to other strains, even from the same species. For example, B.
longum BB536 is claimed to alleviating symptoms of allergy by promoting Th1 response, whereas B. longum ACTT15707 showed a strong induction of interleukin 10 (IL-10) and could help in the treatment and prevention of gastrointestinal infection (Sanz et al., 2007). Recently,Srutkova et al. (2015)have shown thatB.longumssp.longumCCM 7952, but notB.longumssp.
longum CCDM 372, protected mice from the development of experimental colitis suggesting that careful selection might be crucial in providing beneficial outcome in clinical trials with probiotics bacteria.
Our understanding of bifidobacteria-host interactions, their symbiosis and how bacteria are able to differentially modulate the host immune response is growing, but it is still in its infancy. Little is known about the molecular mechanism of this relationship and the specific bacterial components responsible for the beneficial effect are generally unrecognized. The immune effects of probiotics can be exerted directly by live microbial cells, but also by bacterial cell components localized either in the cell wall or membrane fraction or secreted compounds.
Bifidobacteriumproduces plenty of cell-associated molecules which may play a key role in host-microbiota interactions i.e., polysaccharide antigens which may contribute in bacterial adherence to the host cells (Salazar et al., 2009; Fanning et al., 2012), pili-like structures mediate bacterial colonization (Turroni et al., 2013) or a serpin-like protease inhibitor taking part in host- microbe interaction in the gut (Turroni et al., 2010). Extracellular proteins produced by bifidobacteria are known to be either released into environment or surface-attached. They may be responsible for the enhancement of the mucosal barrier and immunomodulation due to the possibility of direct interaction with host epithelial or immune cells (Dylus et al., 2013). Some other proteins, such as cytosolic proteins: metabolic enzymes,
housekeeping, ribosomal proteins and molecular chaperones known as moonlight protein can be also found to be involved in immune cells stimulation, interactions with other bacteria or adhesion phenomena (Henderson and Martin, 2014). It has been demonstrated that in addition to compounds released to culture supernatants, which have clearly an immunomodulation effect (Hoarau et al., 2006), also DNA from different probiotic strains led to cytokine induction (Lammers et al., 2003). However, still the studies on the immunoreactivity of theBifidobacteriumcell components are scarce.
The aim of this study was to identify immunoreactive proteins of two Bifidobacterium strains: B. longum ssp. longum CCM 7952 and CCDM 372 and compare the efficacy of six different methods used for proteins isolation. The immunoreactivity of proteins was tested by SDS-PAGE and Western blotting using sera from mice mono-colonized with studied strains, sera from rabbits immunized against whole bacteria of studied strains and non-immune human sera. As a result, we have found several major immunoreactive proteins recognized by human sera and an immunogenic proteins which elicit antibody response in mice or rabbit immune system. We also indicated that two methods, namely Heilmann method and extraction by LiCl are usefulness to isolate the immunoreactive proteins.
MATERIALS AND METHODS
Bacterial Strains and Culture Conditions
Bifidobacterium longum ssp. longum CCDM 372 and Bifidobacterium longumssp.longumCCM 7952 were originally isolated from feces of breast-fed healthy child. Both strains were provided by Culture Collection of Dairy Microorganisms (Milcom, Czech Republic). The isolates were cultivated in MRS medium (Difco) supplemented with 0.05% L-cysteine- hydrochloride (Millipore) at 37◦C in anaerobic conditions (GasPak EZ Anaerobe Container System BD, USA) for 48 h.
Lactobacillus paracsei LOCK 0912 for sera preparation was isolated form feaces of healthy newborn and cultivated in MRS medium (Difco) at 37◦C in microaerophilic conditions for 48 h. Cells were harvested by centrifugation (6000 g for 15 min) (Heraeus Contifuge Stratus, Thermo Scientific, Germany) and washed two times in PBS buffer (phosphate buffered saline pH 7,4). StrainB.longum ssp.longum CCM 7952 is characterized by high amount of slime production, therefore, before protein isolation, the culture was centrifuged at 14,500 g several times to get rid of salts and bacterial polysaccharides.
Serum Samples
Mouse Non-immune and Immune Sera
Mouse serum was obtained from germ-free mouse (GF) and gnotobiotic mice mono-colonized withB. longum ssp.longum CCM 7952 orB. longumssp.longumCCDM 372 strains. Germ- free BALB/c mice were kept under sterile conditions in Trexler- type plastic isolators, exposed to 12:12-h light-dark cycles and supplied with autoclaved tap water and 50 kGy irradiated sterile pellet diet Altromin 1410 (Altromin, Lage, Germany)ad libitum.
Fecal samples were weekly controlled for microbial molds and yeast contamination by standard microbiological methodology.
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Górska et al. Immunoreactive Proteins ofBifidobacterium
Eight-week-old GF mice were colonized with a single dose (2 × 108 CFU) of freshly grown Bifidobacterium strains in 200 µl of sterile PBS by intragastric administration. Stability of colonization was checked by plating of feces on MRS agar supplemented with l-cysteine hydrochloride (0.5 g/l) and CFU were counted after anaerobic cultivation for 48 h at 37◦C. Animal experiments were approved by the Animal Experimentation Ethics Committee of the Institute of Microbiology of the Academy of Sciences of the Czech Republic and conducted in accordance with the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (CETS No.: 123).”
Rabbit Immune Sera
Rabbit sera were obtained from Termond White rabbit immunized with bacterial mass ofB.longumssp.longumCCM 7952 (R-7952), B.longum ssp. longum CCDM 372 (R-372) or L. paracsei LOCK 0912 (R-0912) as described before (Górska et al., 2016). The experiments were approved by the 1st Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland (number 12/2012).
Human Sera
Human adults sera were obtained from healthy volunteers from Military Blood Donors Centre in Wroclaw (Poland, pooledn= 10), whereas human umbilical cord sera from healthy women (pooledn=10) were obtained from the Obstetric Clinic of the Medical University of Wroclaw and human adults sera from patients withClostridium difficileinfection (pooledn=10) were obtained from 4th Millitary Hospital in Wrocław. The use of sera samples was approved by the Medical Ethics Committee of the Medical University of Wroclaw (number KB-882-2012 and KB- 631/2015) and was conducted in accordance with the Helsinki Declaration of 1975. Samples were obtained with patients’ written informed consent.
Protein Isolation
Six different methods for protein isolation were tested. The Bifidobacteriumcell pellets (1 g) were suspended in 5 ml of:
I. 10 mM EDTA (Sigma-Aldrich; 30 min., 45◦C) according to McCoubrey and Poxton (2001),
II. 0,2 M glycine (Sigma-Aldrich) pH 2,2 (30 min, RT) according toWright et al. (2005),
III. 1 M LiCl (Sigma-Aldrich; 30 min, RT) according toMcCoy et al. (1975),
IV. 8 M urea (Sigma-Aldrich; 30 min, RT) according toMcCoy et al. (1975),
V. 5 M guanidine hydrochloride (Sigma-Aldrich; 2 h, RT) according toPoxton and Byrne (1981),
VI. 0.5 M Tris-HCl (Serva; pH = 6,8)/SDS (Serva; 0,08%
w/v)/glycerol (POCh; 20% v/v)/1 mM β-mercaptoethanol (Sigma-Aldrich) (v/v) and boiled for 5 min according to Heilmann et al. (1996).
After centrifugation (6000 ×g for 5 min), proteins were precipitated from the resulting supernatant using 3 volumes of cold 95% ethanol (POCh). Followed by overnight incubation
at 4◦C, the precipitated proteins were centrifuged (12,000×g), dissolved in water and dialyzed for 48 h. Protein concentration was measured using the Lowry’s method (Lowry et al., 1951). The prepared protein samples were stored in 1 mg aliquots at−70◦C.
SDS-PAGE and Western Blot Conditions
Equal amounts of proteins samples (10µg) were analyzed on SDS-PAGE using 5–12.5% gels according to Laemmli (1970) using Tris-Glycine-SDS as running buffer and run for about 2 h at 100 V (Mini-Protean Tetra Cell 165–8001; Bio-Rad, USA). After staining with Coomassie Brilliant Blue (Serva) or soaked in transfer buffer (10 mM Tris-HCl, 150 mM glycine, 20% methanol (POCh) pH 8.3) for 30 min, gels were transferred to a polyvinylidene difluoride membrane (Millipore) for immunoblotting (for 1 h at 100 V). Following transfer, the membrane was blocked with 1% of bovine serum albumin (BSA, KPL), dissolved in phosphate buffered saline, and then incubated with selected sera in 1% BSA for 2 h at RT. We used different dilutions of sera but the most effective was:
for rabbit sera 1:5000, mouse sera 1:1000 and for human sera 1:500 (reproducible results, without the Hook effect). After washing in PBS containing 0.25% Tween 20 (PBS-T, Institute of Immunology and Experimental Therapy, PAN) for several times, captured antibodies against protein were detected by incubating the membranes with alkaline phosphatase-labeled goat anti-mouse/rabbit/human IgG antibody (Sigma-Aldrich) diluted 1:10,000. Finally, the membranes were washed as six times with PBS-T, and visualized with solution containing nitro blue tetrazolium (NBT, Roth), 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Roth), and MgCl2 (POCh) for 5 s. Image acquisition (exposure time 1–4 min) was performed using VersaDoc Imaging System (Bio-Rad).
Protein Identification
Protein identification was performed as previously described by us (Górska et al., 2016). Briefly, the immunoreactive protein were separated and purified using Prep-Cell apparatus (Model 491 Bio-Rad). Individual spots were cut out from gels and submitted to tryptic digestion, and analyzed by mass spectrometry (spectrometer LC-MS/MS Orbitrap, Thermo). Mascot (Matrix Science, London, UK, http://www.matrixscience.com) and statistical analysis were used to identify proteins from peptide mass fingerprints. All searches were performed against the database for Bifidobacterium species. Immunoreactivity of separated proteins was proved using immunoblotting.
RESULTS
To investigate the immunoreactive proteins ofB.longumCCM 7952 (CCM 7952) andB.longumCCDM 372 (CCDM 372) and to identify the differences between these strains we analyzed and compared the profile of reactive proteins obtained with six different methods. The concentration of the protein in extracts varied profoundly and depended on methods and tested strain (Table 1). Generally, we obtained proteins from CCDM 372 with higher yield than from CCM 7952 strain. The protein extracts were run on SDS-PAGE gels and representative results are shown
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