Ni-NTA Agarose Research Articles

Expression of bovine rotavirus VP8 and preparation of IgY antibodies against recombinant VP8.

Group A rotavirus is the leading cause of acute gastroenteritis in cattle and swine. Although, vaccination against this virus is an effective strategy for prevention, additional strategy to control disease is necessary. Egg yolk immunoglobulin (IgY)-based passive immunization could be a better option in preventing this disease. Bovine rotavirus (BRV) is group A rotavirus and possesses a genome of 11 segments of double-stranded RNA. The outer layer of capsid is composed of two proteins (VP7 and VP4), which induce virus neutralizing antibodies. Trypsin cleavage of VP4 produces VP8 (28 kDa) and VP5 (60 kDa) fragments. Since a number of studies have demonstrated the induction of neutralizing antibodies using VP8 subunit vaccines, we have produced IgY against the recombinant VP8. The cDNA spanning the VP8 subunit was amplified from bovine rotavirus-infected cells and cloned into pET21d(+) expression vector to generate recombinant VP8. The resulting carboxy-terminal His-tagged VP8 proteins were expressed in Escherichia coli strain BL21(DE3) by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified protein was used as the immunizing agent to produce polyclonal antibodies in chicken. The resulting polyclonal antisera specifically recognized VP8 in Western blot assay and were able to neutralize BRV replication in cell cultures. These results demonstrate that IgY can be used in immunological assays and, in addition, in passive immunization of newborn calves against BRV.

Evaluation of the immunogenic property of NT H. influenzae protein D with Neisseria meningitidis OMV in BALB/c.

Identifying ideal non typeable Haemophilus influenzae (NTHi) vaccine candidates has not been easy due to extensive sequence and antigenic variation among gene products interacting with the immune system. Protein D (PD) is a highly conserved 42 kDa surface lipoprotein available in all H. influenzae, including NTHi. In this study, the gene encoding PD was cloned from H. influenzae and expressed in Escheriachia coli TOPO10 cell in pBAD vector. Arabinose was used to express recombinant protein. In order to purify the protein, Ni-NTA agarose was used to perform affinity chromatography. Purified PD and PD mixed with outer membrane vesicle (OMV) and alum adjuvant were used for subcutaneous immunization in BALB/c mice. After vaccination, IgG responses to PD-OMV, PD-alum, and PD alone were determined by enzyme-linked immunosorbent assay (ELISA). The recombinant PD containing His6 residues showed a molecular weight of 42 kDa. Anti-PD IgG was detected after first immunization in all groups of mice compared to the negative control group, and it increased after first vaccination, but results showed that the addition of OMV to PD led to a remarkable increase in IgG responses. Our results suggest an important role for OMV as an adjuvant and show how it could potentially be used when conjugated to H. influenzae PD or other safe subunit vaccine candidates.

Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

Development of serological procedures for sensitive, rapid detection of Cucumber mosaic virus in Lilium

Cucumber mosaic virus (CMV) causes severe agricultural losses in Korea and has a very large host range. To produce virus-free plants, it is necessary to develop efficient, sensitive virus detection methods. In this study, we collected leaf samples from Lilium showing characteristic symptoms of CMV infection from Gang-won Province, Korea in 2011 and amplified the coat protein (CP) gene from CMV from the samples by RT-PCR. Three CMV isolates were found to belong to subgroup IA based on the nucleotide sequence of the CP gene. We cloned the complete CP gene into the pET21d(+) expression vector to generate recombinant coat proteins. After expressing His-tagged coat proteins in Escherichia coli strain BL21 (DE3) by IPTG induction, we purified the proteins using Ni-NTA agarose beads and used the purified CMV recombinant CP for antiserum production. The resulting polyclonal antisera specifically recognized CMV from infected lily samples, as determined by indirect enzyme-linked immunosorbent assay (ID-ELISA) and Western blotting assays. In addition, we developed an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay using the polyclonal antiserum to enable sensitive, reliable, cost-effective detection of CMV. This study demonstrates that three CMV isolates belong to subgroup IA, and that CMV-specific PAbs can be used to detect CMV in epidemiological studies.

Fiber optic biosensor for transdermal glucose based on the glucose binding protein

In previous work, a painless, noninvasive method of collecting transdermal glucose (TG) passively diffusing through the skin was developed. The transdermal glucose collected in this way has concentrations in the μM range requiring a reliable sensor that can measure glucose at these levels. In this study, a fiber optic biosensor for μM glucose based on the glucose binding protein (GBP) is described. The GBP was labeled with BADAN in the H152C position and immobilized on Ni-NTA agarose beads via metal-histidine interaction. The portable, low-cost biosensor system consists of an optical fiber with the sensitized beads trapped on one end, and appropriate optics and electronics on the other end. The control software and the visual interface for the optical sensor is designed and implemented in LabVIEW and runs on a tablet computer. Optimization of sensor response was performed by varying the amount of Ni-NTA-GBP sensitized beads and its distance to the optical fiber. The biosensor exhibited a stable response to buffer and in 10.0μM glucose even after ∼16h of continuous use. Glucose response is reversible when cycled between phosphate-buffered saline solution and various glucose solutions. The response time of the biosensor in 6μM glucose was approximately 50s. A linear relationship (r2=0.990) between sensor response and glucose concentrations from 4.00 to 20.00μM was obtained. Measurement of transdermal glucose diffusion was demonstrated with Yucatan minipig skin as surrogate for human skin. The results show the potential of this fiber optic biosensor as a transdermal glucose monitoring system at the point of care.

THE EXPRESSION AND PURIFICATION OF OCTA-ARGININE APOPTIN AND ITS ABILITY TO KILL CANCER CELLS

<p><strong>Objective: </strong>In this research, <em>chicken anemia virus</em> apoptin optimized genetically for expression in <em>Escherichia coli</em> and also modified using (His)<sub>6</sub> tag, (Arg)<sub>8</sub> tag, and HlyA tag intended for purification needs, penetration enhancement, and secretion from<em> </em>bacterial host<em> </em>to the growth media.</p><p><strong>Methods</strong><strong>:</strong><strong> </strong>The modified apoptin gene was optimized using an Integrated DNA Technology (IDT). The gene (606 bp) then ordered and synthesized by Eurofins. The apoptin gene was expressed using <em>E. coli</em> BL21 CodonPlus as host, in cultivation temperature of 37 °C, and 25 °C and purified using Ni-NTA agarose beads. The addition of (His)<sub> 6</sub> tag enabled the apoptin to be purified in only one step by using nickel column. The expression and purification data analyzed qualitatively as well as quantitatively using SDS-PAGE. MTT assay was used to identify the antitumor effect of octa arginine-apoptin to two kinds of cancer cells, cervix HeLa cancer cell and colon Widr cancer cell. The viability of cell was analyzed when the cell incubated in the variation concentration protein for 72 h.</p><p><strong>Results: </strong>The constructed apoptin gene were expressed in <em>E. coli </em>successfully. The MTT assay indicated that Octaarginin-Apoptin was able to induce apoptosis of HeLa and Widr cells lines in a dose-dependent manner. The recombinant apoptin without tagging with octa-arginine, have no ability to induce apoptosis of HeLa and Widr cells lines. <strong></strong></p><p class="Abstract"><strong>Conclusion: </strong><em>This octa arginine-apoptin may in the future allow the development of a therapeutic protein that is able to kill cancer cells specifically.</em></p>

Cloning, expression, and purification of recombinant major mango allergen Man i 1 in Escherichia coli

In recent years, the number of people around the world who suffer from fruit allergies has increased. Mango can induce anaphylaxis, and two major mango allergens have been identified – Man i 1 and Man i 2. Apart from their molecular weights and pI values, no other information about them is known. This work identifies the DNA and amino acid sequences of Man i 1 and constructs an expression system for recombinant Man i 1 (rMan i 1). Firstly, 3′ and 5′ RACE assays were used to identify the cDNA fragment of Man i 1. Subsequently, the full length of Man i 1 cDNA was inserted into a pET-21a(+) vector, and the inserted plasmid was transformed to Escherichia coli BL21 (DE3) to express rMan i 1. The conditions for the expression of rMan i 1, including IPTG concentration, induction temperature, and induction time, were optimized. The highest amount of soluble rMan i 1 was obtained after induction with 0.1 mM IPTG at 16 °C for 20 h. The His-tagged rMan i 1 was purified using Ni-NTA agarose and its identity was verified using an anti-histidine antibody and the serum of a mango-allergic person. Additionally, rMan i 1 was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and shared 86.2% identity in amino acid sequence of GAPDH from wheat. Finally, an E. coli expression system of rMan i 1 was established, with the potential to be used in immunotherapy against mango allergy or the development of assays for detecting the residues of mango allergens.

Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L.intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L.intermedia venom.

Cloning, expression, and characterization of a novel sialidase from Brevibacterium casei.

The sialidase gene from Brevibacterium casei was cloned in pET28a and overexpressed as a histidine-tagged protein in Escherichia coli BL21(DE3). The histidine-tagged sialidase protein was purified and characterized from the crude cell extracts of isopropyl-β-d-thiogalactopyranoside-induced cells using Ni-NTA agarose chromatography. SDS-PAGE using the purified sialidase indicated a single band at 116 kDa. This sialidase showed maximum activity at a pH of 5.5 and temperature of 37°C. The kinetic parameters Km and Vmax for the artificial substrate 2'-(4-methylumbelliferyl)-α-d-N-acetyl-neuraminic acid sodium salt hydrate were 1.69 × 10-3 mM and 244 mmol·Min-1 ·mg-1 , respectively. The sialidase may catalyze the hydrolysis of terminal sialic acids linked by the α-(2,3) and α-(2,8) linkage of polysialogangliosides, but it does not act on monosialotetrahexosylganglioside (GM1), which offers it a great potential for commercially producing GM1 from polysialogangliosides.

Development of a label-free SPR sensor for detection of matrixmetalloproteinase-9 by antibody immobilization on carboxymethyldextran chip

Surface plasmon resonance (SPR) immunosensor has been widely utilized for monitoring antigen-antibody interactions. The sensor measures changes of refractive index upon binding of analyte molecules to specific ligand immobilized on the sensor chip. This effort reports development of SPR immunosensor for real-time and label-free detection of recombinant human matrix metalloproteinases-9 (MMP-9), which has been associated with malignant tumor progression and metastasis by matrix degradation. MMP-9 was expressed in Escherichia coli BL21 and purified by Ni-NTA agarose column. CMD 50 D was activated by EDC/NHS for immobilization of monoclonal anti-MMP-9. Atomic force microscopy images showed uniform distribution of anti-MMP-9 over the sensor chip. Equilibrium constant (KD), maximum binding capacity (Rmax) and ∆Gb values for interaction of MMP-9 and anti-MMP-9 were 0.4nM, 680 µRIU and −53.51kJ/mol, respectively. Concentration of MMP-9 in saliva samples was determined, with linearity in the range of 10–200ng/mL. The limit of detection was found to be 8pg/mL, being lower than most of the previously reported techniques.

Cloning and characterization of filamentous temperature-sensitive protein Z from Xanthomonas oryzae pv. Oryzae.

The ftsZ gene from Xanthomonas oryzae pv. Oryzae was amplified by PCR with the specific primers, and the recombinant plasmid pET-22b-ftsZ was constructed successfully. The FtsZ with a 6× His tag was overexpressed in a soluble form in Escherichia coli BL21 and purified through a Ni-NTA agarose column. The purified recombinant FtsZ showed a single band on SDS-PAGE with an apparent molecular mass of about 44 kDa, and confirmed by western blotting analysis. The optimum temperature for GTPase activity of the recombined FtsZ was 50 °C, and the optimum pH was 7.0. The recombinant FtsZ showed good stability and retained >95 % activity at 50 °C for 240 min. The GTPase activity followed Michaelis–Menten kinetics with the KM of 1.750 mM and the Vmax of 0.155 nmol Pi/min/nmol FtsZ respectively.

Cleavage of fusion proteins on the affinity resins using the TEV protease variant

It is documented that the tobacco etch virus protease (TEVp) variant TEVp3M is less efficient in cleaving the fusion protein bound to Ni-NTA resin at relatively low temperature. Here, we determined that, using the GFP fusion substrate bound to Ni-NTA or Strep-tactin agarose, activity of the TEVp5M variant was higher than that of the other TEVp construct, and about 15% higher than that of the TEVp3M. The GST fusion proteins immobilized on Strep-tactin agarose or Glutathione Sepharose were efficiently cleaved by purified TEVp5M at specified conditions using GFP reporter for visual track and detection. After on-column cleavage of three fusion constructs using the cognate TEVp5M constructs, two target proteins with relatively high purity were separated from Ni-NTA or Amylose agarose. With elution of the buffer containing 1 M NaCl, maize sulfiredoxin was released from Ni-NTA resin via on-column cleavage. Our results confirmed that TEVp5M efficiently cleaved the fusion proteins bound to the four affinity matrices. By combination with appropriate affinity handles, the cognate TEVp5M mediating tag removal enabled purification and cleavage of the fusion proteins, removal of the protease, and separation of the target proteins from the affinity resin to be accomplished in one step.

Impact of mutations in DNA gyrase genes on quinolone resistance in Campylobacter jejuni.

Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance-determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild-type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni-NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP-dependent DNA supercoiling activity. A quinolone-inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone-resistant C. jejuni. An analysis of the structure-activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R-1 and a chloride substituent at R-8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin-resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP-resistant C. jejuni. Copyright © 2016 John Wiley & Sons, Ltd.

Structure prediction, expression, and antigenicity of c‐terminal of GRP78

Glucose-regulated protein 78 (GRP78) is a typical endoplasmic reticulum luminal chaperone having a main role in the activation of the unfolded protein response. Because of hypoxia and nutrient deprivation in the tumor microenvironment, expression of GRP78 in these cells becomes higher than the native cells, which makes it a suitable candidate for cancer targeting. Suppression of survival signals by antibody production against C-terminal domain of GR78 (CGRP) can induce apoptosis of cancer cells. The aim of this study was in silico analysis, recombinant production, and characterization of CGRP in Escherichia coli. Structural prediction of CGRP by bioinformatics tools was done and the construct containing optimized sequence was transferred to E. coli T7 shuffle. Expression was induced by isopropyl-β-d-thiogalactoside, and recombinant protein was purified by Ni-NTA agarose resin. The content of secondary structures was obtained by circular dichroism (CD) spectrum. CGRP immunogenicity was evaluated from the immunized mouse sera. SDS-PAGE analysis showed CGRP expression in E. coli. CD spectrum also confirmed prediction of structures by bioinformatics tools. The enzyme-linked immunosorbent assay using sera from immunized mice revealed CGRP as a good immunogen. The results obtained in this study showed that the structure of truncated CGRP is very similar to its structure in the whole protein context. This protein can be used in cancer researches.

Expression of HCV Alternative Reading Frame Protein (Core+1/F) in Baculovirus Expression System and its Evaluation for Assessment of Specific Anti-core+1 Antibody in Iranian HCV Infected Patients.

Hepatitis C virus (HCV) genome contains an overlapping reading frame which results in alternative core protein (ARFP). Baculovirus expression system was used as a powerful eukaryotic vector system to express core+1/F protein for the first time. This recombinant core+1/F protein was used to assess the anti-core+1 antibody in anti-HCV drug resistant and sustained virologic response (SVR) patients. The core+1 coding sequence from HCV genotype 1 was designed and synthesized in pUC57 vector. It was subcloned into baculovirus donor plasmid pFastBacTM HTA and transposed into baculovirus shuttle vector (bacmid) to transfect Sf9 cells. Recombinant core+1 protein was purified using Ni-NTA agarose under native condition and verified using SDS-PAGE electrophoresis and Western blotting. An enzyme-linked immunosorbent assay (ELISA) was developed using this purified protein to assess anti-core+1 antibody in 28 anti-HCV drug resistant patients and in 34 patients with sustained virologic response (SVR) in comparison with 31 healthy volunteers used as the negative control. Expression of HCV core+1 protein in Sf9 cells was confirmed by using SDS-PAGE and Western blotting. Antibody titer against core+1 protein in anti-HCV drug resistant patients was significantly higher than that in both the healthy volunteers and SVR patients (p < 0.0001). HCV core+1 protein was expressed successfully in a baculovirus expression system in high yield in order to develop an ELISA to assess the anti-core+1 antibody. Further studies are needed to reveal the potential application of core+1 protein in anti-HCV treatment prognosis.

Cloning, expression and characterization of the gene encoding the enolase from Fusobacterium nucleatum

The gene encoding enolase from Fusobacterium nucleatum (FnENO) was cloned and analyzed for the first time. The gene comprises of 1302 nucleotide base pairs and encodes 433 amino acids. The gene sequence alignment demonstrated the presence of several distinct insertions and deletions, compared with the human enzyme. The gene for recombinant FnENO was inserted into the pLATE 31 vector system and expressed in E. coli BL21(DE3) cells as a soluble protein. The protein was purified by affinity chromatography using a Ni-NTA agarose matrix and shown on SDS-PAGE to be a 46 kDa protein. The molecular weight of the octameric form of the purified recombinant protein was determined as being 375 kDa by size exclusion chromatography. Optimal enzyme activity was observed at pH 8.5 and the enzyme remained stable at a range of different temperatures from 30 to 60°C. Using 2-phosphoglyceric acid as substrate for the purified enzyme, KM, kcat and kcat/KM were determined as 0.48 mM, 20.4 s–1 and 4.22 × 104 M–1s–1, respectively. Potential drug binding sites of FnENO were detected using homology modeling. These data could facilitate the design of new inhibitors of F. nucleatum which has already been shown to be resistant to several known antibiotics.

Inducing Humoral Immune Responses Against Regulatory T Cells by Foxp3-Fc(IgG) Fusion Protein.

The existence of a developed network of suppressory factors and cells against an immune response in different cancers has been proven; regulatory T cells are a typical issue. Therefore their depletion, elimination, or suppression has been assessed in different research studies that were not entirely successful. By applying an improved vaccine against regulatory T cells, we have evaluated the B cell response elicited by the vaccine in an experimental design. A previously described DNA vaccine and recombinant protein of Foxp3-Fc fusion were produced and used in the vaccination regimen. DNA construct and respective protein were injected into C57BL/6 mice. After 2 weeks, serum levels of IgG antibody and its subtypes against Foxp3 were investigated by ELISA. To produce recombinant Foxp3 for ELISA antigen coating, pET24a-Foxp3 vector was transformed into Escherichia coli strain BL21 as host cells. Afterward, protein was expressed and then purified using Ni-NTA agarose. SDS-PAGE and Western blot analysis were carried out to confirm protein expression. The expression analysis of Foxp3 was confirmed by SDS-PAGE followed by Western blot analysis. FOXP3-Fc DNA vaccine/fusion protein vaccination regimen could induce T helper-dependent humoral responses. Due to the effectiveness of Foxp3-Fc(IgG) in inducing humoral responses, it would be expected to be useful in developing vaccines in tumor therapies for the removal of regulatory T cells as a strategy for increasing the efficiency of other means of immunotherapy.

Cloning, Expression, and Characterization of a Thermophilic Endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B.

The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg−1, respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL−1 and 131.75 U·mg−1, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future.

Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni–NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV.

Serodiagnosis of Toxoplasma gondii infection in bovines from Kerala, India using a recombinant surface antigen 1 ELISA

Data on the prevalence of toxoplasmosis in farm animals from India is scanty. Though a few reports exist on prevalence of toxoplasmosis in small ruminants, information on toxoplasmosis in large ruminants is virtually nonexistent from India. An antibody detection recombinant ELISA specific for Toxoplasma gondii was laboratory standardized using recombinant surface antigen 1 (SAG1) protein. A 958-bp truncated sequence coding for tachyzoite stage specific SAG1 protein was amplified and expressed in Escherichia coli BL21(DE3) cells. A high-level expression of the histidine-tagged thioredoxin fusion protein was obtained after 8 h of incubation. The recombinant protein was affinity purified by Ni-NTA agarose chromatography and characterized by SDS-PAGE and Western blot. Subsequently, the diagnostic potential of the recombinant protein was assessed with 258 cattle sera samples from field by a laboratory standardized recSAG1 ELISA. Sera from 71.8% of the cattle showed sero positivity for T. gondii specific IgG. The sensitivity and specificity of the recSAG1 ELISA were 84.38% and 87.88%, respectively in comparison to indirect fluorescent antibody test (IFAT). This is the first report on sensitive serodetection of Toxoplasma infection in bovines from India.