دانلود مقاله ترجمه شده تشخیص هویت آنتی ژن محافظ جدید از Bordetella pertussis – مجله الزویر

فهرست مطالب:

مقدمه
مواد و روش ها
آنالیزهای کامپیوتری
سویه های باکتریایی و شرایط رشد
سلولها
سرم های خونی
کلونینگ مولکولی ژن irp1-3
بیان پروتین نوترکیب
ایمنی سازی موش
تعیین سیتوکینین ها
ELISAها
تست های فاگوسیتوز
آنالیز لکه های وسترن
نتایج
انالیزهای محاسباتی پروتین صادر شده
کلونینگ و تخلیص irp1-3 نوترکیب
بحث

بخشی از ترجمه:

پروتئین های انتی ژنی که بیان انها تحت کمبود شدید اهن القا می شود، یک وضعیت زیست محیطی است که پاتوژن های باکتریایی طی کلونیزاسیون با ان مواجه می باشند که می تواند نامزد های بالقوه برای واکسن های پیشرفته باشد. با استفاده از پروتومیک های ایمنی ما انتی ژن های جدید B pertussis با حداکثر بیان تحت کمبود اهن را شناسایی کردیم. در میان انها Bp1152 واکنش شدید ویژه ای با ihG تخلیص شده از خون افراد آلوده به پرتوزیس یا سیاه سرفه نشان داد. آنالیزهای کامپیوتری حاکی از وجود IRP-3 به عنوان یک پروتین غشایی دیمر دخیل در جذب آهن بود. داده های ازمایشی سطح در معرض این پروتین را اشکار کرده و افزایش آن را تحت کمبود شدیدی اهن مستقل از فاز واگیری باکتریایی نشان داد. ایمنی دادن موش با IRP-3 نوترکیب منجر به واکنش شدید انتی بادی گردید.این انتی بادی ها نه تنها پروتین بومی روی سطح باکتری ها سازماندهی کردند بلکه منجر به افزایش کارایی فاگوسیتوزی باکتری ها با PMN انسانی شد که یک فعالیت کلیدی حفاظتی در برابر پاتوژن ها می باشد. بر همین منوال، IRP1-3 در برابر B.prutussis در موش های مدل عمل حفاظتی نشان داد. بیان IRP1-3 در میان ایزوله های بالینی B.prutussis حفاظتی بود و به طور مثبت با کمبود اهن در این سویه ها تنظیم گردید. با کنار یکدیگر گذاشتن این نتایج می توان گفت که این پروتین می تواند یک کاندید واکسن جدید باشد.

۱ مقدمه

نگرانی ها در خصوص ایمنی ایمنی این واکسن ها منجر به تولید فرمولاسیون های غیر سلولی متشکل از ترکیبی از ان شده است. سیاه سرفه یک بیماری تنفسی حاد در انسان است که توسط B. pertussis و اندک مواقعی توسط B.parapertussis ایجاد می باشد. علی رغم ایمنی جهانی آن، این بیماری هنوز یکی از عومل مرگ و میر در سرتا سر جهان محسوب می شود. با ورود واکسن های سلولی سیاه سرفه در اواسط دهه ۱۹۴۰ این بیماری تا حد زیادی متوقف شد. اگرچه این واکسن ها به طور کلی کارامد می باشند تمام واکسن های سلولی منجر به واکنش های تب و موضعی می شود. نگرانی ها در خصوص ایمنی این واکسن ها منجر به تولید فرمولاسیون های غیر سلولی از جمله توکسین پرتوزیس، پراکتین، هماگلاتین های فیبری و سروتیپ های مختلف شد. طی سال های گذشته علی رغم پوشش بیش از حد واکسنی، وقوع سیاه سرفه در حال افزایش می باشد. حتی در کشورهای پیشرفته پرتوزیس به یک بیماری غیر قابل پیشگیری حتی با واکسن در میان کودگان تبدیل گردیده است. کارایی و اکسن های موجود را می توان با افزودن انتی ژن های B.pertusis بهبود بخشید. تمام ایمنوژن های موجود در فرمولاسیون های سلولی فعلی پروتین های بیان شده در فاز تنظیم مثبت باکتری ها توسط سیستم فسفوریلی Bvg AS می باشند.مطالعات اخیر منجر به ایجاد برخی تردید ها در رابطه با بیان این انتی ژن ها در فنوتیپ های عفونی گردیده اند.

بخشی از مقاله انگلیسی:

 Antigenic proteins whose expression is induced under iron starvation, an environmental condition that bacterial pathogens have to face during colonization, might be potential candidates for improved vaccine. By mean of immune proteomics we identified novel antigens of Bordetella pertussis maximally expressed under iron limitation. Among them, Bp1152 (named as IRP1-3) showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals. Computer analysis showed IRP1-3 as a dimeric membrane protein potentially involved in iron uptake. Experimental data revealed the surface-exposure of this protein and showed its increase under iron starvation to be independent of bacterial virulence phase. Immunization of mice with the recombinant IRP1-3 resulted in a strong antibody response. These antibodies not only recognized the native protein on bacterial surface but also promote effective bacterial phagocytosis by human PMN, a key protecting activity against this pathogen. Accordingly, IRP1-3 proved protective against B. pertussis infection in mouse model. Expression of IRP1-3 was found conserved among clinical isolates of B. pertussis and positively regulated by iron starvation in these strains. Taken together these results suggest that this protein might be an interesting novel vaccine candidate. © ۲۰۱۱ Elsevier Ltd. All rights reserved. 1. Introduction Whooping cough (pertussis) is an acute respiratory illness in humans caused mainly by Bordetella pertussis, and less frequently by Bordetella parapertussis [1]. The disease continues to be a signifi- cant cause of morbidity and mortality worldwide, despite universal immunization [2]. The introduction of whole cell pertussis vaccines in the mid-1940s significantly reduced disease burden. Although generally effective, whole cell vaccines are reactogenic, causing fever and local reactions. Concerns about the safety of these vaccines led to the development and introduction of acellular (Pa) formulations, composed of some combination of B. pertussis antigens including pertussis toxin (PT), pertactin (Prn), filamentous hemaglutinin (FHA), and different fimbriae serotypes [3,4]. Over the last years, despite high vaccine coverage the incidence of whooping cough has been increasing [5–۷]. Even in developed countries, pertussis had become the most commonly reported vaccine-preventable disease among young children. Efficacy of existing Pa vaccines might improve by the addition of further B. ∗ Corresponding author. Tel.: +54 221 4833794; fax: +54 221 4833794. E-mail address: mer@quimica.unlp.edu.ar (M.E. Rodriguez). pertussis antigens. All immunogens included in current acellular formulations are proteins expressed in the virulent phase of the bacteria positively regulated by the BvgAS two-component phosphorelay system [8,9]. Recent studies raised some doubts about the expression of these antigens in the infective phenotype. Modulating conditions in intranasal cavity [10] might eventually induce the temporal lack of expression of one or more of these vaccine antigens in the infecting bacteria. On top of this, recently published data reported the existence of circulating strains of B. pertussis not expressing two of the main vaccine antigens [11]. An effective way to improve pertussis control may comprise updating current vaccines by including antigens that are expected to be present in the infective phenotype. Antigenic proteins whose expression is induced under physiological conditions, i.e. iron limitation, might be a good option. The lack of iron is an environmental stress that human pathogens have to face during infection. Successful microbial pathogens have developed mechanisms to overcomehostironrestriction,includingproductionandutilization of low-molecular-weight iron chelators (siderophores), capture of siderophores produced by other organisms, and direct removal of iron from host proteins via specific bacterial cell surface receptors [12]. Because these factors are essential for bacterial survival in vivo they gain importance as potential targets for the development of vaccines and therapeutic agents. In order to gain a better insight into the iron starved phenotype of B. pertussis and the immunogenicity of the differentially expressed proteins we used proteomic tools in combination with complete genome sequences to relate genome-wide expression response of this pathogen to iron starvation [13]. The results showed that iron restriction induces the expression of proteins of different functional classes, some of them immunogenic. Among the latter, Bp1152, a protein identified in the outer membrane subproteome, named IRP1-3 by us, showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals [13]. In this study we show that IRP1-3 is a surface-exposed highly immunogenic protein expressed under physiological conditions and protective against infection in mouse model. We further show that its gene is conserved among circulating strains and it is expressed under iron limitation irrespective of the virulence state of the bacteria. Altogether these results suggest that this protein is a good candidate for further study as vaccine antigen. 2. Materials and methods 2.1. Computational analysis Sequence similarity searches were performed to identify close homologous proteins to IRP1-3 using BLAST. The proteins were retrieved with E-values below 1 × ۱۰−۴, and aligned using CLUSTALX [14]. To search for templates to build structural models, fold assignment methods as HHpred [15] and FFAS03 [16] were applied. The best templates obtained were used to build models using the program Modeller [17]. The quality of the models was assessed using PROSA II [18]. TMHMM [19], and DAS [20] servers were used to estimate putative transmembrane regions. 2.2. Bacterial strains and growth conditions B. pertussis strains BP536, a streptomycin-resistant derivative of Tohama I, and BP537, a Bvg-phase locked derivative of Tohama I [21] were used in this study. B. pertussis strain BP536 was transformed with plasmid pCW505 [22] (kindly supplied by Dr. Weiss, Cincinnati, OH) which induces cytoplasmic expression of GFP without affecting growth, or antigen expression [22]. The clinical strains used for PCR and western blot analysis were isolated from Argentinean patients in the period 2002–۲۰۰۷٫ B. pertussis strains were cultured as previously described [13] with a few modifications. Briefly, bacterial strains were grown in Bordet Gengou agar (BGA) plates and further subcultured in Stainer-Scholte (SS) for 24 h. Bacterial cells were harvested by centrifugation, washed with sterile iron-free saline solution. Equal volumes of bacterial cell suspensions were used to inoculate 100 ml of iron-replete SS (36 M iron) (SS), and iron-depleted SS (without addition of FeSO4·۷H2O) (SS-Fe), with or without 50 mM of MgSO4. Bacteria were cultured at 37 ◦C under shaking condition for 20 h, and subcultured twice in the respective culture media. Iron-depleted medium was prepared as previously described [13]. The chrome azurol S assay [23] was used to monitor the presence of siderophores in culture supernatants of B. pertussis grown in iron-depleted medium. E. coli BL21-CodonPlus (DE3)-RIL transformed with plasmid pET28/His6-IRP1-3 was grown in Luria Bertani (LB) medium at 37 ◦C supplemented with kanamycin at a final concentration of 50 g/ml and used in expression experiments. 2.3. Cells Peripheral blood neutrophils (PMN) were isolated from heparinized venous blood using Ficoll-Histopaque (Sigma, St. Louis, MO) gradient centrifugation. PMN were harvested and remaining erythrocytes removed by hypotonic lysis. PMN purity determined by cytospin preparations exceeded 95%, and cell viability was 99% as determined by trypan blue exclusion. Prior to functional assays, PMN were washed twice with DMEM supplemented with 0.2% of BSA (Sigma, St. Louis, MO), resuspended, and used immediately. All experiments described in this study were carried out with freshly isolated PMN lacking FcRI (CD64) expression, as monitored by FACS analysis with anti-FcRI mAb 22 (BD Biosciences, San Diego). 2.4. Sera Immunoglobulin G (IgG) fractions from pooled sera of pertussis patients with high titers against B. pertussis (as measured by ELISA [24]) were obtained using protein G (Pharmacia Biotech, Uppsala, Sweden) chromatography as described before [25]. 2.5. Molecular cloning of irp1-3 gene The full-length coding region of irp1-3 gene of B. pertussis was amplified from genomic DNA by polymerase chain reaction (PCR) using Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA). The PCR reaction was carried out using the following conditions. Initial denaturation cycle at 94 ◦C for 5 min, followed by 35 cycles of 94 ◦C for 1 min, 56 ◦C for 30 s, 68 ◦C for 1 min, and a final extension of 5 min at 68 ◦C. The primers for PCR cloning were as follows: forward primer 5 -agc gtc gac tga tga aga aag cct tgc tac-3 and reverse primer 5 -act aag ctt tca gta ccc gcc ctt ctt g-3 . The inserted restriction sites are underlined. The PCR product corresponding to the full-length gene sequence of irp1-3 was cloned into the SalI and HindIII restriction sites of the pET28a (Novagen, Madison, WI) expression vector to generate the recombinant protein with an Nterminal histidine tag. The cloned PCR fragment was sequenced using single primer extension to confirm that no PCR-induced mutations had been introduced (Macrogen Inc., Seoul, Korea). 2.6. Recombinant protein expression To express the recombinant protein (rIRP1-3), transformed E. coli strain BL21-CodonPlus (DE3)-RIL cells were grown at 37 ◦C in LB medium (500 ml) to an A600 of 0.4. Protein expression was induced by adding isopropyl -d-1-thiogalactopyranoside (IPTG), to a final concentration of 0.5 mM and incubating for 3 h. The bacterial pellet was harvested and resuspended in denaturing buffer containing 50 mM Na3PO4, 300 mM NaCl, 10 mM imidazole and urea 8 M (pH 7.4). The extract was then clarified by centrifugation at 10,000 × g for 20 min and the recombinant protein in the supernatant was purified by cobalt-nitrilotriacetic acid-agarose affinity resin under denaturing conditions according to the supplier’s instructions (Pierce Biotechnology, Rockford). After unbound proteins were washed from the column, the His-tagged recombinant protein elution was carried out in 150 mM imidazole and protein purity was analyzed on 12.5% (w/v) sodium dodecyl sulphatepolyacrylamide gel (SDS-PAGE) stained with Coomassie blue. The purified protein (rIRP1-3) was dialyzed, lyophilized and reconstituted in phosphate buffered saline (PBS). 2.7. Mice immunization Three to 6 weeks old female BALB/c mice were used in animal experiments. The mice were obtained from and bred in the specific pathogen-free breeding rooms of the animal facility of the Faculty of Veterinary, University of La Plata. A group of 8 female BALB/c mice were immunized intraperitoneally (i.p.) with 4 g of rIRP1-3 protein emulsified in complete Freund’s adjuvant (FA), or Alum (Al). A booster dose was given on J. Alvarez Hayes et al. / Vaccine 29 (2011) 8731–۸۷۳۹ ۸۷۳۳ the 21st day with 4 g of rIRP1-3 emulsified in incomplete Freund’s adjuvant or Alum, respectively. Another two groups of mice (n = 8 each group) were i.p. immunized with 5 × ۱۰۶ cells of heatinactivated Bp−Fe (wIDP) or Bp+Fe (wIRP) emulsified withAlum.As negative controls, two separated groups of 8 mice were i.p. immunized with an equal amount of adjuvant alone or PBS. Since there was no significant difference in immune responses between animals immunized with FA or PBS, the data of animals immunized with PBS are not shown here. The mice were bled at days 0, 21 and 36 and the serum was separated by centrifugation and stored at −۲۰ ◦C until analysis. Animal handling and all the experimental procedures were carried out in compliance with the recommendations of the “Guide for the Care and Use of Laboratory Animals” of the National Research Council (Academy Press, 1996, Washington).