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Identification of Epitope in EAV N Protein

Identification of a novel conserved B cell epitope in the N protein of EAV (Bucyrus strain)
Running title: Identification of epitope in EAV N protein.
Highlights:
One EAV N-specific mAb 1C11 was developed.
A minimal linear peptide epitope within the N protein was identified.
The identified epitope was conserved among different regional EAV strains.
The mAb and identified epitope may be useful diagnostic tools for EAV infection.
Abstract
Objective: To identify the minimal epitope of N protein of the equine arteritis virus (EAV).
Methods: The full-length sequence of EAV N gene was cloned by RT-PCR and ligated into pET32a vector for expression. The recombinant pET-N protein was expressed in E. coli and purified by Ni affinity chromatography. The purified N protein was used to immunize mice for preparing monoclonal antibody (mAb). The reactivity of mAb was evaluated by Western blot and immunofluorescence assay (IFA). The peptides were identified using the prepared mAb by indirect ELISA and Western blot. The homology analysis was performed using DNAMAN software.
Results: Recombinant EAV N protein was successfully expressed in the procaryon expression system. An EAV N-reactive mAb was selected and designated as 1C11. Indirect ELISA results showed that overlapping domain of MBP-N10 and MBP-N11 was recognized by the mAb 1C11. Further, the indirect ELISA and Western blot showed that 101QRKVAP106 was the minimal linear epitope of the EAV N protein. The homology analysis showed that the identified epitope is conserved among all EAV isolated strains, with the exception of the ARVAC which is a modified live virus vaccine strain.
Conclusion: One EAV N-specific mAb was developed and a minimal linear peptide epitope within the N protein was identified. The EAV N-specific mAb and the defined linear peptide epitope of EAV N protein may be useful for the development of specific diagnostic tools and design of vaccine.
Keywords: Epitope; N protein; Equine arteritis virus; Monoclonal antibody
Introduction
Equine arteritis virus (EAV) is the etiologic agent of equine viral arteritis (EVA) which is a respiratory and reproductive disease of horses [1-3]. EAV was first isolated from horses in Ohio in 1953. It is the prototype virus of the family Arteriviridae (genus Arterivirus, order Nidovirales) [4, 5]. EAV infection of horses has been reported in many countries including New Zealand, Australia, and South Africa [6-10].
EAV is a positive-sense, enveloped and single-stranded RNA molecule with a length of 12.7kb [11]. It contains two large open reading frames (ORFs, 1a and 1b) and seven smaller ORFs (2a, 2b, and 3 to 7). ORFs 1a and 1b encode two replicase polyproteins (pp1a and pp1ab), whereas the ORFs 2a, 2b, 5, 6, and 7 encode the known EAV structural proteins E, GS, GL, M, and N, respectively [12]. Moreover, ORFs 3 and 4 encode glycosylated membrane-associated proteins whose functional role is still under debate [13, 14].
EAV N can be used as an alternative protein candidate of diagnostic antigens and accounts for 35-40% of the total virion protein [15]. B cell epitopes involved in the immune response against EAV [16]. In the present study, we aimed to identify the precise B cell epitope using a monoclonal antibody (mAb) against EAV N protein. Our result will provide important information for developing serological diagnosis of EAV infection and understanding the antigenic structure of EAV N protein and vaccine design.
Materials and methods
Ethics statement
Care and use of laboratory animals and all animal experiments were in accordance with animal ethics guidelines established by the Institutional Animal Ethics Committee in China. All animal studies were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (SYXK (H) 2006-032).
Cell lines and virus
SP2/0 myeloma and Rabbit kidney 13 (RK-13) cells were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) in a humidified 5% CO2 atmosphere at 37°C. All culture media were supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, Invitrogen) and antibiotics (0.1mg/ml streptomycin and 100 IU/ml penicillin).The Bucyrus strain of EAV (GenBank accession No. NC-002532.2, a highly cell culture-adapted strain provided by the key laboratory of Tropical and Subtropical Animal Viral Diseases in Yunnan province, China) was propagated in RK-13 cells and stored at -80℃.
Expression and characterization of recombinant EAV N protein
The full-length sequence of EAV N gene was cloned by RT-PCR using the following primers: 5?-CCCGGATCCATGGCGTCAAGACGATC-3? (upstream) and 5?-TTTGTCGACTTACGGCCCTGCTGGAGGCGCAAC-3? (downstream). The primers contained BamH I and Sal I restriction sites (italicized). The purified and digested PCR product was ligated into an expression vector pET32a (Novagen, Germany). The pET-N recombinant plasmid was transformed into E. coli BL21 (DE3) and 1mM isopropyl-?-D-1-thiogalactopyranoside (IPTG, Invitrogen, USA) was used for inducing expression of N protein. The recombinant proteins were obtained from the bacterial lysates. The insoluble inclusion bodies were washed and solubilized with phosphate buffered saline (PBS, pH 7.4). Then, the recombinant N protein fused with 6 His-tags was evaluated by SDS-PAGE and purified by Ni affinity chromatography according to manufacturer’s instruction (Invitrogen).
Preparation and characterization of mAbs against N protein
EAV N-reactive mAb was generated as previously described [17]. Briefly, 6-week-old female BALB/c mice were immunized with the purified recombinant N protein (100?g per mouse) mixed with an equal volume of Freund’s complete adjuvant (FCA, Sigma, USA). Two booster injections containing the same amount of purified N protein in an equal volume of Freund’s incomplete adjuvant (FICA) were given at 2-week intervals. The purified N protein without adjuvant was injected intraperitoneally as the final immunization. After three days of the final injection, the mice were euthanized and their splenocytes were fused with SP2/0 myeloma cells using polyethylene glycol (PEG4000, Sigma). The hybridoma cells were seeded into 96-well plates and selected in hypoxanthine-aminopterin-thymidine (HAT) selection medium (DMEM containing 20% fetal bovine serum, 100g/ml streptomycin, 100IU/ml penicillin, 100mM hypoxanthine, 16mM thymidine, and 400 mM aminopterin). After 5 days, the medium was removed and replaced with hypoxanthine-thymidine (HT)-DMEM medium (DMEM containing 20% fetal bovine serum, 100g/ml streptomycin, 100IU/ml penicillin, 100mM hypoxanthine, and 16mM thymidine). After selection in HAT and HT medium, hybridoma supernatants were screened for evaluating reactivity and specificity of mAb by Western blot and immunofluorescence assay (IFA). The class and subclass of the mAb was determined using a SBA ClonotypingTM System/HRP (Southern Biotechnology Associates, Inc., Birmingham, AL35260, USA).
Polypeptide design and expression
Eleven overlapping peptides spanning the N protein were designed (Table 1,). For each peptide, a pair of oligonucleotide strands was synthesized. Each pair of oligonucleotide strands was annealed and cloned into the BamHâ… and Sal I sites of pMAL™-C4x vector and expressed as MBP-N fusion proteins. These MBP-fused proteins were named consecutively MBP-N1 to MBP-N11. The recombinant plasmids were transformed into E.coli Rosetta (DE3) (Novagen). Each MBP-fused polypeptide was induced by IPTG and screened by indirect ELISA. Briefly, MBP tags and purified N protein were used as negative and positive controls, respectively. Ninety-six-well microtiter plates were coated with expressed MBP-N fusion proteins at 4℃ overnight and blocked with 5% skim milk for 1 h at 37℃. After washing three times by PBST (PBS plus 0.5% Tween-20), 100 ?l of mAb was added to wells and incubated at 37℃ for 1 h. Then, the plates were washed three times by PBST and incubated with diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam, UK) at 37℃ for 1 h. The color was developed and the reaction was stopped with 2M H2SO4. The absorbance at 450 nm was measured. All assays repeated three times and the average of the three values was calculated.
Identification of the epitopes
The MBP-N-fusion proteins were identified by indirect ELISA and Western blot using the mAb. Indirect ELISA was performed as mentioned above. For Western blot, the purified MBP-N recombinant proteins were electrophoresed on SDS-PAGE, and then transferred to a nitrocellulose membrane. Nonspecific antibody binding sites on the membrane were blocked with 5% skim milk in PBS overnight at 4℃. The membrane was washed and incubated with mAbs for 1h at 37℃. The membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody after five times washing with PBST. Following another five times washing, the color was developed using 3,3-diaminobenzidine (DAB) and terminated by rinsing the membrane with deionized water.
Homology analysis
To evaluate the conservation of the identified linear epitope among EAV from different geographic areas, the identified epitope and the corresponding regions of other regional EAV virus strains were aligned using DNAMAN software (Lynnon BioSoft Inc., USA).
Results
Production of recombinant EAV N protein and mAb
As shown in Fig.1a, Recombinant EAV N protein was successfully expressed in the procaryon expression system. A clear single target band with expected molecular weight was displayed. Accordingly, the recombinant EAV N protein was suitable as an antigen for immunization and hybridoma screening.
Purified proteins were utilized to immunize BALB/c mice. After cell fusion and selection, an EAV N-reactive mAb generated from one hybridoma cell line was selected for its strong reactivity against N protein. This mAb was designated as 1C11. As shown in Fig.1b, c, mAb 1C11 reacted with recombinant N protein and total protein of EAV (Fig.1b, c). The reactivity of mAb was also assessed using RK-13 cells by IFA (Fig.1d). The mAb only reacted with EAV infected cells and not reacted with uninfected control RK-13 cells.
Identification of EAV N epitope
To localize linear antigenic epitopes within the N protein, 11 16-amino acid long MBP fused peptides (MBP-N1 – MBP-N11) were expressed and probed by mAb 1C11 by indirect ELISA. The results showed that MBP-N10 (91TVSWVPTKQIQRKVAP106) and MBP-N11 (95VPTKQIQRKVAPPAGP110) epitopes were recognized by the mAb 1C11 (OD450 > 1) (Fig. 2a). All the left fragments (MBP-N1-9) failed to react with the mAb. Because adjacent epitopes have 12 overlaps, we deduced that the linear epitope located in the overlapping domain of MBP-N10 and MBP-N11 (95VPTKQIQRKVAP106). To identify the minimal linear peptide epitope within this overlapping domain, a series of truncated polypeptides were expressed as MBP-fusion proteins. Ultimately, the indirect ELISA and Western blot showed that 101QRKVAP106 was the minimal linear epitope for the reactivity of the EAV N protein recognized by mAb 1C11 (Fig. 2c, d).
Homology analysis
Sequence alignment was performed to evaluate the conservation of the identified epitope among different regional EAV viruses (Fig. 3). The identified epitope is conserved among all EAV isolated strains, with the exception of the ARVAC which is a modified live virus vaccine strain.
Discussion Mapping location of viral protein epitopes and defining the degree of their conservation may play an important role for understanding of the antigenic structure, virus-antibody interactions. It may be very useful for vaccine design and clinical applications. In this study, the B cell epitopes of EAV N protein were identified using a mAb.
Epitope mapping using mAbs has become a powerful tool to study protein structure and provides new tools to diagnose diseases and design vaccines [18]. Here, we defined one peptide epitope of EAV N protein in by using an EAV N-specific mAb. To our knowledge, epitope on the N protein of EAV has been published, but no previous studies about 101QRKVAP106 have been reported. Starick et al. [19] produced a mAb against the N protein to detect EAV. Weiland et al. [20] used the same method to produce a mAb against the N protein of EAV and to distinguish different virus isolates from semen and tissue samples after passaging through RK-13, Vero and fetal equine kidney cells. However, the minimal epitope of these mAb was not defined precisely. Similar to the work of Starick et al. and Weiland et al. [19, 20], a mAb named 1C11 against EAV N protein was prepared by using recombinant N protein expressed in E. coli and used for identifying B-cell epitopes on EAV N protein. mAb 1C11 reacted well with EAV by WB and IFA, thus this antibody may be a useful detection tool in EAV diagnosis.
mAbs are useful and effective for mapping antigenic epitopes of viral proteins. In this study, for epitope mapping, 11 overlapping peptides from EAV N protein were expressed with MBP tags and identified by ELISA to screen linear epitopes. The ELISA results showed that the epitope located in the sharing region of MBP-N10 (91TVSWVPTKQIQRKVAP106) and MBP-N11 (95VPTKQIQRKVAPPAGP110). Then this region (95VPTKQIQRKVAP106) was expressed, and 7 peptides with deletions were obtained to identify the precise epitope. According to the results of ELISA and Western blot, 101QRKVAP106 was considered as the minimal linear epitope of EAV protein. This result is different from the previous studies [15, 21] which stated that the precise epitope of N protein located in amino acids 1-69. This may be due to the difference of the specificity and reactivity of the mAbs. Sometimes, a mAb can react with different locations of a viral protein.
Sequence alignment showed that the identified epitope is very conservative among distinct regional EAV strains, but with a mutation of one amino acids on the ARVAC N protein epitope. This result suggests a slight regional difference emerged in this epitope. Therefore, it is possible to distinguish anti-Bucyrus EAV antibody from anti- ARVAC EAV antibody by using the epitope as antigen. This will be helpful in distinguishing the distinct regional EAV infection. This finding indicates that the N epitope of EAV identified in our study have a potential use in serological monitoring and differential diagnosis.
In conclusion, one EAV N-specific mAb was developed and a minimal linear peptide epitope within the N protein was defined. The EAV N-specific mAb and the defined linear peptide epitope of EAV N protein may be useful for the development of specific diagnostic tools and design of vaccine.

Isolation of Casein from Milk and Powdered Milk

1.1 Introduction
(Walsh, 2002) stated that, proteins are biological macromolecules composed of amino acids proteins consist of one or more polypeptide which are the chain of amino acids interconnected by peptide bonds.Alberts et al., (2013) detailed that, amino acids of proteins is either hydrophobic or hydrophilic in nature. Therefore the resulting polypeptide chain shows an amphipathic characteristic. Hydrophilic amino acids exist peripherally in some biological system and they are highly water soluble. Whereas some amino acid does not exist the polar groups to the environment.
‘’The most important factors that influence protein solubility are structure, size, charge and the solvent‘’ (Burgess, and Deutscher, 2009). Also Burgess, and Deutscher (2009) stated that, once the precipitation obtained, the solution can be separated by centrifugation or precipitation.
‘’Protein precipitates are aggregates of protein molecular large enough to be visible and to be collected by centrifugation. The distribution of hydrophilic and hydrophobic residues at the surface of a protein determines its solubility properties‘’. (Rosenburg, 2006). Precipitation is mainly done for concentrate the target protein. And it is attained by adding reagents such as salts (ammonium sulfate) or organic solvents (acetone or ethanol). (Hatti-Kaul and Mattiasson, 2003)
1.1.1 Isolation of casein
Milk contains three kinds of proteins: caseins, lactalbumins, and lactoglobulins, all of which are globular proteins. (Spurlock, 2014). Ahluwalia and Dhingra, (2005) stated that, Casein is a combination of phosphoproteins presenting in milk and cheese.it is existing to the amount of 3% in milk along with 4-5% of lactose and 3-4% of fats and the rest is water. Caseins exist in micelles which are composed of sub micelles linked by the characteristic of hydrocolloid which are freely suspended in the aqueous phase of milk. (Tarte, 2009). Casein can be electrophoretically fractioned into four major components: alpha-, beta-, gamma-and kappa- casein. Casein develops precipitation from milk at pH 4.6, which has a negative charge when compare to the pH of the milk. Therefor it can be precipitate as salt by adding acids. (Miller, Jarvis and McBean, 2006).
1.2 Objectives
To learn the methods of protein precipitation and to relate the solubility of protein with its structure.
To learn the methods of isolation of casein from milk and to determine the percentage of casein presented in the (powdered) milk.
1.3 Materials
Test tubes
Beakers
Pipette
Clamp
Filtering paper
Electronic balance
Watch glass
Bunsen burner
Albumin sample
Ammonium sulfate
Sodium hydroxide
Copper sulfate
Ethanol
Picric acid
Lead nitrate
Powdered milk
Warm water
1.4 Methodology
1.4.1 Precipitation by salts
Albumin, 3.00ml was taken into a test tube, ammonium sulfate was added to it and was mixed until the solution gets saturated. The solution was allowed to stand for about 5 minutes and filtered by using filter paper. The biuret test was done to the filtered solution. 3.00 ml of filtered solution was taken into another test tube and same amount of NaOH was added to it, CuSO4 was added drop by drop.
1.4.2 Precipitation by organic solvents
Albumin, 1.00 ml was taken into a test tube using a pipette. And 4.00 ml of ethanol was added .the solution was mixed well and was allowed to stand.
1.4.3 Precipitation by acidic agents
Picric acid solution, 1.00 ml was added into 1.00ml of albumin solution.
1.4.4 Precipitation by heavy metal ions
Lead nitrate, 8 drops were added into 1.00 ml of albumin.
1.4.5 Precipitation by heat and acid
Albumin, 10 ml was taken into a test tube and the upper part of the solution was held over the Bunsen flame. After the observation few drops of 1% acetic acid were added.
1.4.6 Isolation of casein
Powdered milk (non-fat), 17.5 g was weighed by using electronic balance and was dissolved by adding 62.5 ml of warm water in a 200ml beaker. Acetic acid (10%) was added in a drop wise manner with stirring until the liquid changes in to clear solution. the obtained solution was filtered by using clamp, filtering material and beaker. The yield casein was allowed to dry and was weighed using electronic balance. Biuret test was done for the filtered solution. 3.00 ml of filtered solution was taken into another test tube and same amount of NaOH was added to it, CuSO4 was added drop by drop.
1.5 Results
Test Observation Interference Precipitation by metal ions
White color precipitation
Proteins can be precipitated by metal ions(positive for proteins)
Precipitation by heat and acid
Initially cloudy white precipitation was observed on the upper part of the solution and by adding acetic acid white color precipitation was observed.
Proteins can be precipitate by heat and acid(positive for proteins)
Precipitation by organic solvents
White color precipitation was observed
Proteins can be precipitate by organic solvents(positive for proteins)
Precipitation by acidic agent
White color precipitation was observed
Proteins can be precipitate by acidic agents. (positive for proteins)
Precipitation of salts
Biuret test
White color precipitation was observed.
Purple color ring was observed

Proteins can be precipitate by salts.
Positive for proteins.
Isolation of casein
Biuret test for filtration
Casein 13.01g was weighed
Purple color ring was formed in filtered casein solution
Yield %= × 100
= × 100
= 74.30%
Positive for proteins.
1.6 Discussions
Precipitation of protein can be obtained by isoelectric precipitation method. ‘’isoelectric precipitation is the most widely used method’ (Fox and McSweeney, 2003). Proteins can be precipitated by bringing their pH to its isoelectric point in which protein solubility is very low. (Shankara, 2008)
Proteins can be precipitate by salts in two ways, half saturation with ammonium sulfate and full saturation with ammonium sulfate. Rashmi, (2002) stated that, different proteins show different precipitation reaction towards diverse agents. The full saturation with ammonium sulfate was done in the laboratory. Also the filtrate was tested by biuret reagent, resulted purple color. ‘Compounds with two or more peptide bonds give a violet color with alkaline copper sulfate’ (Rashmi, 2002)
Proteins are strong in solution when they are enclosed by entirely hydrogen-bonded water molecules, as water molecules with additional hydrogen bonding ability have greater entropy and are more aggressive. (Chaplin, 2014) hydrated sphere decrease the non -polarity. Higher the diameter of the sphere higher the solubility. For an example, it is easy to precipitate globulin from proteins by adding salts, than albumin because globulin has small diameter of hydrated sphere when compare to albumin.
The similar concept is used in precipitating proteins by organic solvents and acidic agents. Organic solvents remove the hydrated sphere and decrease solubility resulting increase precipitation. Acids neutralize the polarity of the hydrated sphere and decrease solubility in order to increase precipitation.
Denaturation occur on heating or adding acidic agents to proteins. Therefor its change the polarity of a protein by changing the arrangements of polar and non-polar groups within the molecule. Less polarity decrease the solubility and increases the precipitation.
Precipitation by heavy metal ions lead nitrate was used instead of lead acetate or mercuric nitrate. Shankara(2008) stated that, metal ions which are positively charged interrelate with negatively charged groups of the protein producing precipitation as metal-proteinate complex.
According to the percentage of yield and from the result of biuret test of the filtrate, there can be some proteins present in the filtrate. Because, the milk contains about 3.5% protein by weight and of the total protein, about 80% is casein and 20% is whey protein. (Miller, Jarvis and McBean, 2006)
Filtration of casein can be done in two ways. Such as, gravitational filtration and sucktional filtration.
1.7. Conclusion
Proteins were precipitated by using metal ions, heat, organic solvents, acidic agents and salts.
The percentage of yield casein of the sample is 74.30%.
References Ahluwalia, V. and Dhingra, S. (2005). College Practical Chemistry. [Online] Google Books. Available at: http://books.google.lk/books?id=1OgRECl_nwMC

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