Anexo 8. Proteínas de ovario solubles (tabla 3)

Las proteínas de ovario solubles fueron analizadas en:La Universidad de Columbia en la Ciudad de Nueva York. En la tabla 3 se muestra la información que se nos otorgó una vez realizado el estudio, cada link proporciona un análisis detallado de cada proteína.

Tabla 3. Proteínas identificadas mediante LC-MS/MS.

Full length article

Identification of immunogenic proteins from ovarian tissue and recognized in larval extracts of Rhipicephalus (Boophilus) microplus, through an immunoproteomic approach

Patricia Berenice Ramírez Rodríguez^a, Rodrigo Rosario Cruz^b, Delia Ines Domínguez García^b, Rodolfo Hernandez Gutierrez^a,

Rodolfo Esteban Lagunes Quintanilla^c, Daniel Ortu~no Sahagún^d, Celia Gonzalez Castillo^d, Abel Gutierrez Ortega^a, Sara Elisa Herrera Rodríguez^a, Adriana Vallejo Cardona^a, Moises Martínez Velazquez^a,*

aCentro de Investigacion y Asistencia en Tecnología y Dise~no del Estado de Jalisco, AC. Av. Normalistas 800, Col. Colinas de la Normal, C.P. 44270, Guadalajara, Jalisco, Mexico

bUnidad de Investigacion en Biotecnología, Salud y Ambiente (BIOSA-UAGro) de la Unidad Academica de Ciencias Naturales de la Universidad Autonoma de Guerrero, Carretera Federal Chilpancingo-Acapulco, Ex-Rancho el“Shalako”, Petaquillas, Guerrero, C.P. 39105, Mexico

cCentro Nacional de Investigaciones en Parasitología Veterinaria-INIFAP, Carretera Federal Cuernavaca Cuautla No. 8534, Col. Progreso, C.P. 62550, Jiutepec, Morelos, Mexico

dInstituto de Investigacion en Ciencias Biomedicas (IICB), CUCS, Universidad de Guadalajara, Sierra Mojada No. 950, Col. Independencia, C.P. 44340, Guadalajara, Jalisco, Mexico

h i g h l i g h t s g r a p h i c a l a b s t r a c t

A classical approach immunizing cattle with total ovarian proteins was followed.

The detection of ovarian proteins was achieved by ELISA plates coated with proteins from larval homogenates.

Several immunogenic proteins such as Vitellogenins and yolk Cathepsin were identified.

Vitellogenin-1 and Vitellogenin-2 transcripts are synthesized in the ovaries ofR. microplus.

Vitellogenins represent attractive candidates for development of anti- tick vaccines.

a r t i c l e i n f o

Article history:

Received 21 April 2016 Received in revised form 3 October 2016 Accepted 6 October 2016 Available online 8 October 2016

a b s t r a c t

Rhipicephalus (Boophilus) microplusticks are obligatory hematophagous ectoparasites of cattle and act as vectors for disease-causing microorganisms. Conventional tick control is based on the use of chemical acaricides; however, their uncontrolled use has increased tSresistant tick populations, as well as food and environmental contamination. Alternative immunological tick control has shown to be partially effective.

The only anti-tick vaccine commercially available at present in the world is based on intestinal Bm86

*Corresponding author. Av. Normalistas 800, Col. Colinas de la Normal, C.P. 44270, Guadalajara, Jalisco, Mexico.

E-mail addresses:[email protected](P.B. Ramírez Rodríguez),[email protected](R. Rosario Cruz),[email protected](D.I. Domínguez García), [email protected](R. Hernandez Gutierrez),[email protected](R.E. Lagunes Quintanilla),[email protected](D. Ortu~no Sahagún),celia_glz@

hotmail.com (C. Gonzalez Castillo), [email protected] (A. Gutierrez Ortega), [email protected] (S.E. Herrera Rodríguez), [email protected] (A. Vallejo Cardona), [email protected](M. Martínez Velazquez).

Contents lists available atScienceDirect

Experimental Parasitology

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / y e x p r

http://dx.doi.org/10.1016/j.exppara.2016.10.005 0014-4894/©2016 Elsevier Inc. All rights reserved.

Experimental Parasitology 170 (2016) 227e235

Keywords:

Tick

Boophilus microplus Ovary

Immunogen Vitellogenin Vaccine

protein, and shows a variable effectiveness depending on tick strains or geographic isolates. Therefore, there is a need to characterize new antigens in order to improve immunological protection. The aim of this work was to identify immunogenic proteins from ovarian tissue extracts ofR. microplus, after cattle immunization. Results showed that ovarian proteins complexed with the adjuvant Montanide ISA 50 V generated a strong humoral response on vaccinated cattle. IgG levels peaked at fourth post- immunization week and remained high until the end of the experiment. 1D and 2D SDS-PAGE- Western blot assays with sera from immunized cattle recognized several ovarian proteins. Reactive bands were cut and analyzed by LC-MS/MS. They were identified as Vitellogenin, Vitellogenin-2 pre- cursor and Yolk Cathepsin. Ourfindings along with bioinformatic analysis indicate thatR. microplushas several Vitellogenin members, which are proteolytically processed to generate multiple polypeptide fragments. This apparent complexity of vitellogenic tick molecular targets gives the opportunity to explore their potential usefulness as vaccine candidates but, at the same time, imposes a challenge on the selection of the appropriate set of antigens.

©2016 Elsevier Inc. All rights reserved.

1. Introduction

Rhipicephalus(Boophilus)microplusticks are obligatory hema- tophagous ectoparasites of cattle and act as vectors for disease- causing microorganisms such as protozoa of the genus Babesia spp. and bacteria Anaplasma spp. (Perez de Leon et al., 2010).

Together, these microorganisms and ticks may induce anemia and weight loss and decrease milk and meat production in the para- sitized cattle, as well as causing the depreciation of cattle skin value in the market. The conventionalR. microplustick control is based on the use of chemical acaricides (Martins et al., 2002). However, uncontrolled use of these agents has increased the incidence of tick populations resistant to acaricides (Crampton et al., 1999; Martins and Furlong, 2001; Klafke et al., 2006; Parizi et al., 2009). Addi- tionally, chemical treatments have severe limitations due to the food safety and environmental pollution problems they cause. The alternative immunological control of tick infestations has shown to be partially effective. Proven vaccines such as Gavac™and Tick- Gard™are based on the recombinant Bm86 antigen, aR. microplus midgut cell membrane protein (Rand et al., 1989; Willadsen et al., 1989; Gough and Kemp, 1993). These vaccines are used to immu- nize cattle in order to induce immunoglobulins that have the ca- pacity to bind the tick's intestinal cells after blood feeding, and subsequently cause the ectoparasite's death (Maritz-Olivier et al., 2012; Rodriguez-Valle et al., 2012).

Currently, the only anti-tick vaccine commercially available in the world is Gavac™, based on Bm86. Its high protection, durability and environmentally friendly characteristics have made it widely used (Rodríguez, 2000). Nevertheless, its effectiveness varies depending on tick strains or geographic isolates due to the varia- tions in Bm86 sequence between different tick populations (García- García et al., 2000). Consequently, research efforts are still being made worldwide to identify new candidate antigens for a more effective vaccine to control tick infestations (de la Fuente et al., 2007). The development of anti-tick vaccines converge in the identification, molecular cloning andin vitroproduction of proteins that play important roles in tick physiology, such as cell signaling, modulation of host immune response, pathogen transmission, embryogenesis, digestion, and intermediary metabolism (Parizi et al., 2009). It is known that a significant portion of immuno- globulins that are taken up with blood meal retain biological ac- tivity during their passage through the tick's gut, hemolymph and salivary glands. Therefore, the potential targets for a protective immune response could be any of the antigens in the internal or- gans that may be in contact with host antibodies, including anti- gens located in ovaries (Wang and Nuttall, 1999). In this context, the aim of this work was to identify immunogenic proteins from

ovarian tissue of R. microplus utilizing an immunoproteomic approach. Additionally, it was hypothesized that sera from immu- nized cattle can recognize proteins present in both larvae and ovaries. Antigens reported here could be potentially used as vaccine targets, which could result in a detrimental effect on oogenesis, embryogenesis and reproductive rates ofR. microplusand, there- fore, are a promising option for addressing the tick population control.

2. Material and methods

2.1. Ticks

Engorged adult female ticks ofR. micropluswere obtained from a susceptible strain reared under laboratory conditions at CENID- PAVET-INIFAP, located in Jiutepec, Morelos, Mexico.

2.2. Tissue dissection and protein extraction

A lysate of total proteins was made from dissected ovarian tissue of 15e20 engorged female ticks, collected the following day after the spontaneous detachment from calves (approximately 21 days after larvae infestation), following the protocol described by Rachinsky et al (Rachinsky et al., 2007). with modifications. 100 mg of ovarian tissue were weighed and placed in an Eppendorf tube and 1 ml of PBS was added. Tissue was disrupted with a Teflon^® conical pestle and then suspension was passed through a syringe (barrel size 1 ml; needle size 29 Gauge x½”) 10 times, to help tissue fragmentation, until it was fully disintegrated. Suspension was incubated at 70C for 5 min and immediately after, at 26C for 5 min; this was repeated three times. Next, lysate was sonicated for 5 min and centrifuged at 13,000 rpm for 15 min at 4C, then the supernatant was separated. Protein samples were quantified by the DC protein assay (Bio-Rad, USA) (Lowry et al., 1951). The absorbance was read at 750 nm by a Microplate Spectrophotometer (XMark™, Bio-Rad, USA).

2.3. One-dimensional electrophoresis

Analysis of the ovarian proteins was carried out using one- dimensional (1D) electrophoresis gels to verify patterns. Briefly, polyacrylamide gels 7 cm wide x 8 cm long and 1 mm thick (12%) were made. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was conducted with Mini-PROTEAN^®Tetra Elec- trophoresis System (Bio-Rad). 10mg of ovarian proteins were dis- solved in SDS-PAGE sample buffer and heated at 100C for 5 min.

Molecular weight markers (Precision Plus Protein Standards, Bio- P.B. Ramírez Rodríguez et al. / Experimental Parasitology 170 (2016) 227e235

228

Rad) were diluted 1:19 in SDS-PAGE sample buffer and heated at 100C for 5 min. Gels were run at 120 V for 80 min in electro- phoresis buffer (192 mM glycine, 25 mM Tris, 0.1% SDS), stained with Coomassie blue and visualized/photographed using a Gel-Doc XR Documentation System (Bio-Rad).

2.4. Bovines

Six animals aged six months, European crossbred with average weight of 140 kg were used. They were kept in experimental animal facilities at CENID-PAVET-INIFAP. The animals were individually housed in isolation pens and were fed on fodder receiving water ad libitum. Animals were randomly assigned to two experimental groups. Thefirst group (four animals) was immunized with total ovarian proteins, whereas the second group (two animals) only with PBS plus adjuvant as a control.

2.5. Immunization

The experimental protocol was approved by the Institutional Committee of Animal Experimentation of CENID-PAVET-INIFAP.

Cattle were injected subcutaneously in the neck with 2 ml/dose (100mg of total ovarian proteins) using a 5 ml syringe and an 18G needle. Emulsion was made with ovarian protein lysate in PBS plus adjuvant Montanide ISA 50 V (anhydromannitolether- octodecenoate in mineral oil) (Seppic, Paris, France) at 1:1 vol ratio.

Two immunizations were applied; thefirst immunization on the first day and the booster immunization after 15 days (Fig. 1). All the animals were checked regularly for any signs of local reaction or clinical abnormalities post immunization and received treatment in accordance with the animal experimentation rules described in the Guide for Care and Use of Laboratory Animals for the CENID-PAVET- INIFAP.

2.6. Blood collection

Blood samples were collected from cattle every week for 10 weeks. Three samples of 7 ml blood were collected into sterile tubes using vacutainer syringe needles (0.838 mm) from caudal vein. Samples were maintained at 4C until arrival at the labora- tory. Serum was separated from cellular components by centrifu- gation at 5000 rpm for 1 min before being stored at 20 C.

Antibody titers were determined using an antigen-specific indirect enzyme-linked immunosorbent assay (ELISA).

2.7. ELISA test

Total protein lysate from larvae was prepared according to the method of Shahein et al. (Shahein et al., 2008). Briefly, one gram of 6-day-old larvae was homogenized in ice-cold phosphate-buff- ered saline (PBS),filtered, sonicated, and centrifuged at 15,000g for 20 min at 4C. Supernatant was separated and protein con- centration quantified by the DC protein assay (Bio-Rad) as mentioned above. 1 mg of total proteins was diluted in 100 ml carbonate buffer (50 mM Na2CO3) and used to coat a 96 well microplate, and then incubated overnight at 4C. The plate was washed four times with 300ml washing buffer (PBS/0.5% Tween 20), then blocked with blocking buffer (PBS/0.5% Tween 20 and 5%

low fat milk) during one hour at room temperature (25C). Next, the incubation plate was washed with washing buffer as mentioned above. Immunized and non-immunized calves’sera were added by triplicate into wells at a 1:100 dilution ratio. The plate was washed as described above. A secondary antibody (anti- bovine IgG-Phosphatase-Coupled) was used at a 1:2000 dilution ratio. The plate was once more washed and then 200ml of sub- strate paranitrophenyl phosphate (PNN) (Sigma 104) was added.

The plate was incubated for 30 min and read at 405 nm in a

Fig. 1.Antibody response in immunized cattle. IgG levels were determined by ELISA in sera from cattle immunized with the ovarian proteins or adjuvant/saline control. Antibody titers are expressed as the O.D. 405 nm value for the serum dilution 1:100. Arrows indicate the immunization times.

P.B. Ramírez Rodríguez et al. / Experimental Parasitology 170 (2016) 227e235 229

Microplate Spectrophotometer (XMark™, Bio-Rad, USA).

2.8. Two-dimensional electrophoresis

For 2D electrophoresis, immobiline dry-strips gel with linear pH 3e10 and 7 cm in length (Bio-Rad) were rehydrated in gel overnight with 12mg of ovary proteins in 125ml of rehydration buffer (7 M urea, 2 M thiourea, CHAPS), 1 M DTT, and 20 mM IPG Buffer (3e10).

The first dimensional separation was performed using an Ettan IPGphor™ 3 Isoelectrofocusing (IEF) system (GE Healthcare); the program was run with four steps: Step 1: (Step) 300 V/2:30 h; Step 2: (Gradient) 1000 V/0:30 h; Step 3: (Gradient) 5000 V/1:30 h, and Step 4: (Step) 5000 V/0:35 h. After IEF running, the immobiline dry- strips gel containing the focused ovary proteins was equilibrated following two incubation steps: with 1% DTT (w/v), and with 2.5%

iodoacetamide (w/v) for 20 min, respectively. The immobiline dry- strips gel was then transferred onto a 12% polyacrylamide slab gel and 2D separation was carried out under the same running con- ditions as mentioned above for one-dimensional electrophoresis.

Separated proteins were visualized by Coomassie blue staining.

Two-dimensional electrophoresis images were captured using the Gel-Doc XR Documentation System (Bio-Rad) and analyzed by Sigma Gel Software in order to calculate the molecular weight of the proteins. 2D electrophoresis was performed in quintuplicate by sample.

2.9. Western blot assays

1D and 2D immunoblots were performed according to the protocol developed by Towbin et al (Towbin et al., 1979). and Hawkes et al (Hawkes et al., 1982). with some modifications. In brief, total ovary proteins were separated in 12% SDS-PAGE gels and transferred to nitrocellulose membranes using a Mini Trans-Blot apparatus (Bio-Rad) in transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, 20% methanol) at 110 V for 60 min. After blotting, the membrane was blocked in Tris-buffered saline (TBS) containing 0.1% (v/v) Tween 20 (TBS-T) and 5% low fat milk, for 4 h at room temperature. The blot was briefly washed in TBS-T, then incubated with sera from control, pre-immune or immunized animals in a 1:50 dilution in TBS-T overnight at 4C. Following the incubation, the membrane was washed 55 min with TBS-T and incubated for 4 h at room temperature with a 1:2000 dilution of goat anti-bovine IgG coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., PA, USA). The membrane was then washed three times with TBS-T. Detection was carried out using the reagent 4- chloro-1-naphthol (4CN, Bio-Rad).

2.10. LC-MS/MS analysis

The proteins recognized by bovine's sera in nitrocellulose membrane were obtained from the corresponding replicate gel and analyzed by LC-MS/MS. Gel spots were transferred to pre-digested tubes and 100ml 0.01 M DTT/0.1 M Tris, pH 8.5 was added. The tube was placed in a heating block at 55C for 1e2 h. After cooling the tube to room temperature, the liquid was removed and replaced with 100 ml 0.03 M iodoacetamide/0.1 M Tris, pH 8.5. This was allowed to react for 30 min in the dark after which the liquid was removed and the gel was washed as described below.

Gel pieces were prepared for digestion by washing twice with 200ml 0.05 M Tris, pH 8.5/30% acetonitrile for 20 min with shaking, and once with 100ml acetonitrile for several minutes until the gel was opaque white. After removing the acetonitrile, the gel pieces were dried for 20e30 min in a Speed-Vac concentrator.

The gel pieces were digested by adding 0.10mg modified trypsin (sequencing grade, Roche Molecular Biochemicals, Indianapolis, IN)

in 50ml 0.025 M Tris, pH 8.5, or enough volume to completely hydrate the gel. The tubes were placed in a heating block at 32C and left overnight.

Peptides were extracted with 250ml 50% acetonitrile/2% TFA and the combined extracts were reduced in volume in a Speed-Vac concentrator and brought up to 20ml with 0.1% formic acid. 5ml aliquots were analyzed by LC-MS/MS.

LC-MS/MS analysis was done on a Waters Q-Tof Ultima hybrid quadrupole/time-of-flight mass spectrometer with a nano- electrospray source. The eluant was introduced into the source through a 15mm i. d. New Objective PicoTip. Capillary voltage was set at 1.8 kV and cone voltage 32 V; collision energy was set ac- cording to mass and charge of the ion, from 14eV to 50eV. Chro- matography was performed on an LC Packings HPLC with a C18 PepMap column using a linear acetonitrile gradient withflow rate of 200 nl/min.

Raw datafiles were processed using the MassLynx ProteinLynx v.4.1 software and. pklfiles were submitted for searching atwww.

matrixscience.com using the Mascot algorithm. De novo sequencing was accomplished by manual inspection of either the raw MS/MS spectra or spectra processed with the MaxEnt 3 algo- rithm contained in the Waters MassLynx 4.1 software package.

2.11. RNA isolation and cDNA synthesis

100 mg of ovarian tissue were dissected, weighed and placed into an Eppendorf tube and 1 ml of TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) was added to extract total RNA. cDNA was synthesized from 5mg of RNA using the Superscript^®III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions.

2.12. Endpoint polymerase chain reaction

R. microplusVitellogenin 1, Vitellogenin 2 and Vitellogenin 3 encoding sequences were obtained from the GenBank genetic sequence database (GBBR01000071.1, GBBR01000108.1 (Vg-3 in this study) and GBBR01000106.1 (Vg-2 in this study), respectively).

The following primers (Integrated DNA Technologies, Coralville, IA, USA) were used: F-RmVg15⁰-CCA ACT CGC TCA AGA CCT TCe3⁰and R-RmVg15⁰-AAA CCA GGG GTT CCT TGC AC-3’; F-RmVg2(3)5⁰-CTA CCT AAG GCG CGA GAT TGe3⁰and R-RmVg2(3)5⁰-TGG ATT CCG AGA TGT CCT TCe3’; F-RmVg3(2)5⁰-AGC TTC GAC AAG ACC CTC AA-3⁰ and R-RmVg3(2)5⁰-GGT GCT GGT GGT GTA GAG GTe3⁰, using cDNA from ovaries as template. PCR conditions were 94C for 5 min followed by 35 cycles, each consisting of denaturation at 94C for 30 s, annealing at 55C for 30 s, and extension at 72C for 60 s. A final extension step at 72C for 7 min was included. The amplified products were run on 1% agarose gels and analyzed on a UV transilluminator (BioRad Laboratories, Philadelphia, PA, USA).

Control amplification was carried out using the following primers:

F-5⁰- CCC ATC TAC GAA GGT TAC GCC-3⁰and R-5⁰-CGC ACG ATT TCA CGC TCA Ge3⁰, designed fromR. microplus actin(GenBank accession number AY255624.1). PCR conditions were the same as mentioned above.

2.13. Bioinformatic analysis

All primers were designed with the public Primer3 software v.4.0.0 (http://primer3.wi.mit.edu/ (Rozen and Skaletsky, 2000);).

Phylogenetic analysis was performed using the CLC Main Work- bench 6.0 software (CLC bio, Cambridge, MA, USA), with 1000 boostrap replicates.

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3. Results

3.1. Antibody titers in vaccinated cattle

In this work, we sought to explore the ovaries as a potential source of new vaccine candidates tofight cattle ticks. To this end, we dissected ovarian tissue from engorged adult female ticks and disrupted it to obtain a suspension of total ovarian proteins (Fig. 2A). This sample was complexed with the adjuvant Montanide ISA 50 V and used to immunize cattle. Ovarian proteins generated a strong humoral immune response in vaccinated cattle. Antibody titers increased after each immunization, peaked at the fourth week and remained high until the end of the experiment (Fig. 1).

Samples from control animals did not show significant changes in IgG levels. Since sera with the highest IgG levels were found on the fourth week, they were utilized for 1D and 2D western blot assays.

3.2. Detection of immunogenic proteins by western blot assays Sera collected on the fourth post-immunization week were used as thefirst antibody against ovarian proteins run on 1D SDS-PAGE and immunoblots were developed. Some proteins were recog- nized with sera from immunized cattle but not from control or pre- immune sera (Fig. 2BeD).

In order to obtain a higher resolution on the protein recognition, we performed 2D SDS-PAGE-Western blot experiments and found that sera from immunized cattle detected several ovarian proteins (Fig. 3). The reactive spots observed on the nitrocellulose mem- brane were identified in the gel replicate and the corresponding spots were manually cut out with new sterile micropipette ends, collected in sterile Eppendorf tubes, digested with trypsin, and analyzed by LC-MS/MS. Samples were named as they were cut out from the gel as OvaP1, OvaP2, OvaP3, OvaP4a and OvaP4b (Table 1).

3.3. Identification of immunogenic proteins by LC-MS/MS

Tryptic peptides from samples were analyzed using sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS).

Proteins were identified as follows: samples OvaP1 and OvaP2 were identified as Vitellogenin, sample OvaP3 as Vitellogenin-2 precur- sor and samples OvaP4a and OvaP4b as Yolk Cathepsin (Table 1).

4. Discussion

Considerable efforts have been undertaken worldwide to iden- tify and characterize tick molecules for the development of novel or improved anti-tick vaccines. At present, however, there is only one anti-cattle tick vaccine commercially available in the world, which is based on the Bm86 antigen and is marketed under the trade name Gavac™. Past efforts had been focused mainly on gut and salivary gland antigens, and more recently on other internal organs as the ovaries, which are essential for tick reproduction. For example, Rachinsky et al. reported a differential protein expression in ovaries of uninfected and Babesia-infectedR. microplus ticks.

Among the ovarian proteins that were up-regulated in infected ticks were calreticulin, two myosin subunits, an endoplasmic re- ticulum protein, a peptidyl-prolyl cis-transisomerase (PPIase), a cytochromecoxidase subunit, a glutamine synthetase, and a family of Kunitz-type serine protease inhibitors (Rachinsky et al., 2007).

These findings were additionally supported through an ovarian transcriptome study (Heekin et al., 2013). In the present work, we followed a classical vaccinology approach, using total ovarian pro- tein extracts as immunogens, combined with ELISA, 1D and 2D western blot, and LC-MS/MS analyses. In this discovery phase, we could identify three immunogenic proteins, namely Vitellogenin, Vitellogenin-2 precursor and Yolk Cathepsin. Interestingly, despite its importance,R. microplustick vitellogenesis process has not been studied in detail and vitellogenic proteins are just being discovered.

Fig. 2.Electrophoretic pattern of ovarian proteins and immunodetection in 1D-SDS-PAGE. (A) Total ovarian proteins were run on one-dimensional electrophoresis SDS-PAGE (12%) and stained with Coomassie blue. Gel replicas were transferred to nitrocellulose membranes and incubated separately with control (B), pre-immune (C) or immune sera (D), and Western blot assays were developed. (1) Molecular weight marker; (2) Ovarian proteins. Arrows indicate the reactive bands. Representative 1D immunoblotting is shown.

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