pET28a Vector Research Articles

Subtractive proteomic analysis of antigenic extracellular proteins and design a multi-epitope vaccine against Staphylococcus aureus.

Staphylococcus aureus is a versatile Gram's positive bacterium that can reside as an asymptomatic colonizer, which can cause a wide range of skin, soft-tissue, and nosocomial infections. A vaccine against multi-drug resistant S. aureus, therefore, is urgently needed. Subtractive proteomics and reverse vaccinology are newly emerging techniques to design multiepitope-based vaccines. The analysis of 7290 proteomes (sensitive and resistant strains), five potent nonhuman homologous vaccine targets [(UNIPORT ID Q2FZL3 (Staphopain B), Q2G2R8 (Staphopain A), Q2FWP0 (uncharacterized leukocidin-like protein 1), Q2G1S6 (uncharacterized protein), and Q2FWV3 (Staphylokinase, putative)] were selected. These proteins were absent in the gut microbiome, which further enhances the significance of these proteins in vaccine design. These five virulence-associated proteins mainly have a role in the invasion mechanism in the host phagocyte cells. MHC I, MHC II, and B cell epitopes were identified in these five proteins. Finalized epitopes were examined by different online servers to screen suitable epitopes for multi-epitope based vaccine design. Shortlisted antigenic and nonallergenic associated epitopes were joined with linkers to design 30 variants (VSA1-VSA30) of multi-epitope vaccine conjugates. The antigenicity and allergenicity of all the 30 vaccine constructs were identified, and VSA30 was found to have the highest antigenicity and lowest allergenicity, and hence was selected for further study. Accordingly, VSA30 was docked with different HLA allelic variants, and the best-docked complex (VSA30-1SYS) was further analyzed by molecular dynamics simulation (MDS). The MDS result confirms the interaction of VSA30 with MHC (HLA-allelic variant). Thus, the final vaccine construct was in silico cloned in the pET28a vector for suitable expression in a heterologous system. Therefore, the designed vaccine construct VSA-30 can be developed as an appropriate vaccine to target S. aureus infection. VSA-30 still needs experimental validation to assure the antigenic and immunogenic properties.

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b

Recombinant human interferon α2b (rhIFNα2b) is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C. The current identification test for rhIFNα2b is complex. In this study, an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b. RhIFNα2b was used to immunize an alpaca, which established a phage nanobody library. After five steps of enrichment, the nanobody I22, which specifically bound rhIFNα2b, was isolated and inserted into the prokaryotic expression vector pET28a. After subsequent purification, the physicochemical properties of the nanobody were determined. A semiquantitative detection and rapid identification assay of rhIFNα2b was developed using this novel nanobody. To develop a rapid test, the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips. The developed rhIFNα2b detection assay had a limit of detection of 1 μg/mL. The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products. The principle of this novel assay is generally applicable for the rapid testing of other commercial products, with a great potential for routine use in detecting counterfeit recombinant protein products.

Engineering of the CHAPk Staphylococcal Phage Endolysin to Enhance Antibacterial Activity against Stationary-Phase Cells

Bacteriophage endolysins and their derivatives have strong potential as antibacterial agents considering the increasing prevalence of antibiotic resistance in common bacterial pathogens. The peptidoglycan degrading peptidase CHAPk, a truncated derivate of staphylococcal phage K endolysin (LysK), has proven efficacy in preventing and disrupting staphylococcal biofilms. Nevertheless, the concentration of CHAPk required to eliminate populations of stationary-phase cells was previously found to be four-fold higher than that for log-phase cells. Moreover, CHAPk-mediated lysis of stationary-phase cells was observed to be slower than for log-phase cultures. In the present study, we report the fusion of a 165 amino acid fragment containing CHAPk with a 136 amino acid fragment containing the cell-binding domain of the bacteriocin lysostaphin to create a chimeric enzyme designated CHAPk-SH3blys in the vector pET28a. The chimeric protein was employed in concentrations as low as 5 μg/mL, producing a reduction in turbidity in 7-day-old cultures, whereas the original CHAPk required at least 20 μg/mL to achieve this. Where 7-day old liquid cultures were used, the chimeric enzyme exhibited a 16-fold lower MIC than CHAPk. In terms of biofilm prevention, a concentration of 1 μg/mL of the chimeric enzyme was sufficient, whereas for CHAPk, 125 μg/mL was needed. Moreover, the chimeric enzyme exhibited total biofilm disruption when 5 μg/mL was employed in 4-h assays, whereas CHAPk could only partially disrupt the biofilms at this concentration. This study demonstrates that the cell-binding domain from lysostaphin can make the phage endolysin CHAPk more effective against sessile staphylococcal cells.

Integrated Core Proteomics, Subtractive Proteomics, and Immunoinformatics Investigation to Unveil a Potential Multi-Epitope Vaccine against Schistosomiasis.

Schistosomiasis is a parasitic infection that causes considerable morbidity and mortality in the world. Infections of parasitic blood flukes, known as schistosomes, cause the disease. No vaccine is available yet and thus there is a need to design an effective vaccine against schistosomiasis. Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium are the main pathogenic species that infect humans. In this research, core proteomics was combined with a subtractive proteomics pipeline to identify suitable antigenic proteins for the construction of a multi-epitope vaccine (MEV) against human-infecting Schistosoma species. The pipeline revealed two antigenic proteins—calcium binding and mycosubtilin synthase subunit C—as promising vaccine targets. T and B cell epitopes from the targeted proteins were predicted using multiple bioinformatics and immunoinformatics databases. Seven cytotoxic T cell lymphocytes (CTL), three helper T cell lymphocytes (HTL), and four linear B cell lymphocytes (LBL) epitopes were fused with a suitable adjuvant and linkers to design a 217 amino-acid-long MEV. The vaccine was coupled with a TLR-4 agonist (RS-09; Sequence: APPHALS) adjuvant to enhance the immune responses. The designed MEV was stable, highly antigenic, and non-allergenic to human use. Molecular docking, molecular dynamics (MD) simulations, and molecular mechanics/generalized Born surface area (MMGBSA) analysis were performed to study the binding affinity and molecular interactions of the MEV with human immune receptors (TLR2 and TLR4) and MHC molecules (MHC I and MHC II). The MEV expression capability was tested in an Escherichia coli (strain-K12) plasmid vector pET-28a(+). Findings of these computer assays proved the MEV as highly promising in establishing protective immunity against the pathogens; nevertheless, additional validation by in vivo and in vitro experiments is required to discuss its real immune-protective efficacy.

Evaluation of kinetic stability and anti-staphylococcal activity of recombinant LasA protein produced in Escherichia coli.

Objective(s):Staphylococcus aureus has become a major clinical concern due to the growing prevalence of multi-drug resistant (MDR) strains. Enzybioticts are peptidoglycan hydrolases that are recently introduced as an alternative agent to confront the MDR strains with a more effective mechanism than conventional antibiotics. In this regard, our study aimed to evaluate the kinetic stability of LasA protease as a potent enzybiotic in the specific destruction of the S. aureus cell wall. Materials and Methods:The catalytic domain of the Codon-optimized LasA gene was sub-cloned into pET28a vector, and BL21 DE3 cells were used for protein expression. Recombinant LasA protein was affinity purified by Ni-NTA column and staphylolytic activity of the LasA protein against methicillin-resistant strains was evaluated by disk diffusion and MIC test. The kinetic stability was evaluated in different temperatures during 48 hr. Results:Our results revealed that LasA protein can completely prevent the growth of Methicillin-resistant S. aureus (MRSA) strain and inhibit the examined strain at the amount of 4 µg. furthermore, the catalytic domain of LasA protein can tolerate higher temperatures as well. Conclusion:With regard to the failure of conventional antibiotics in treatment of MRSA infections, novel agents to combat multidrug-resistant strains are needed. The present study shows that LasA protein can eradicate MRSA strains, so it can be promising for the treatment of antibiotic-resistant staphylococci infection. The kinetic stability of LasA has also confirmed the possibility of industrial-scale manufacturing for the subsequent use of the enzyme clinically.

Saccharification of Hazelnut and Rhododendron Biomasses Using β-xylanase from Thermotoga naphthophila

Enzymes can be used in various biotechnological applications due to the easy and cheap production. Since xylanase enzymes are preferred in various industries, researchon this enzyme is extensively being carried out. In this study, the β-xylanase gene was cloned from Thermotoga naphthophila, a thermophilic organism. The expression vector pET21a(+) was expressed in Escherichia coli BL21 (DE3). As a result of the studies, the pH, temperature and IPTG concentration of the enzyme were optimized to obtain highest expression. Dinitrosalicylic acid (DNS) was used to determine sugar content of the enzyme. The molecular mass of the purified β-xylanase enzyme was determined using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular mass of the enzyme was calculated to be 38 kDa. Enzymatic hydrolysis of hazelnut shell, rhododendron branch and rhododendron leaves was performed. Released reducing sugar contents from the enzymatic hydrolysis were calculated as 0.8461 mg mL-1, 0.6976 mg mL-1 and 0.3605 mg mL-1 for hazelnut shell, rhododendron branch, and rhododendron leaf respectively. In conclusion, β-xylanase enzyme can be an effective source for enzymatic hydrolysis to produce fermentable sugars from such biomasses.

CspB and CspC are induced upon cold shock in Bacillus cereus strain D2.

Bacillus cereus D2, a psychrotrophic strain, plays an essential role in the restoration of heavy metal-contaminated soils, especially at low temperatures. However, the cold shock response mechanisms of this strain are unclear. In this study, the cold shock response of B.cereus D2 was characterized; as per the Arrhenius curve, 10°C was chosen as the cold shock temperature. Six cold shock-like proteins were found and temporarily named cold shock protein (Csp)1-6; the respective genes were cloned and identified. Quantitative real-time PCR results showed that csp1, csp2, csp3, and csp6 were overexpressed under cold shock conditions. Interestingly, after cloning the respective encoding genes into the pET-28a (+) vector and their subsequent transformation into E.coli BL21 (DE3), the strains expressing Csp2 and Csp6 grew faster at 10°C, showing a large number of bacteria. These results suggest that Csp2 and Csp6 are the major cold shock proteins in B.cereus D2. Of note, the comparison of amino acid sequences and structures showed that Csp2 and Csp6 belong to the CspB and CspC families, respectively. Additionally, we show that the number of hydrophobic residues is not a determining feature of major Csps, while, on the other hand, the formation of an α-helix in the context of a leucine residue is the most dominant difference between major and other Bacillus and E.coli Csps.

Coated recombinant Escherichia coli for delayed release of β-mannanase in the water-based fracturing fluid

A low-temperature active glycosyl hydrolase family 26(GH26) β-mannanase (Man37) was identified in Bacillus velezensis BS-37 by gene mining. The β-mannanase Man37 contained a signal peptide at the N-terminus. The Man37 and Man37△1−25 (without the signal peptide sequence) coding sequences were successfully expressed in Escherichia coli BL21(DE3) using the pET-28a(+) vector. The specific activity of the purified Man37 and Man37△1−25 with guar gum as substrate was 98.12 and 150.34 U/mg, respectively. Man37△1−25 was superior to Man37 due to its higher specific activity and soluble expression. The temperature and pH range of Man37△1−25 was 20−55 °C and pH5.0–10.0, respectively. The enzyme could effectively reduce the viscosity of fracturing fluid from 6200 to 1000 mPa·s after 10 min at 25 °C, which demonstrates its application potential as a gel-breaker in low-temperature hydrocarbon reservoirs. Uncoated recombinant Man37△1−25 cells could completely reduce the viscosity of water-based fracturing fluid within only 3 h, while was greatly prolonged to 18 h with coating polyethylenimine-glutaraldehyde (PEI-GA). Thus, this study provides an effective solution for premature degradation caused by the addition of water-based fracturing fluid together with a short-acting gel-breaker.

Design of a novel and potent multivalent epitope based human cytomegalovirus peptide vaccine: An immunoinformatics approach

Human Cytomegalovirus (HCMV) is one of the most common causes of congenital infections globally. They infect the fetus and cause severe birth defects such as hearing loss, complications in the eyes, epilepsy, autism, and various other neurological disorders. They are also the leading cause of death in patients with compromised immunity. Due to the prevalence and severity of the virus, the United States National Academy of Medicine has announced the development of the HCMV vaccine as a matter of high priority for public health. Hence, this study is focused on designing a novel potent multi-epitope HCMV vaccine. The envelope glycoproteins B, H, L and M of HCMV were targeted to predict T cell (both Helper and Cytotoxic) epitopes that were non-toxic, highly antigenic, and non-allergenic and could induce interferon-γ production. Further, the selected epitopes were subjected to molecular docking and population coverage analysis. Eventually, using the two best epitopes and adjuvants like RS09, flagellin protein and PADRE sequence a 283 amino acid long vaccine was designed. The vaccine is antigenic, non-allergenic, stable, and soluble. The 3D model of the vaccine was developed and validated using the Ramachandran plot. Docking with Toll-like receptor 5 (TLR5) molecule and molecular dynamics simulation confirmed the stability of the vaccine. In silico cloning simulation using pET28a (+) vector showed that the vaccine can be successfully cloned and expressed in E. coli K12 strain. However, the efficacy and the safety of the vaccine need to be further validated by in vivo studies.

Characterization of recombinant E. coli expressing a novel fucosidase from Bacillus cereus 2–8 belonging to GH95 family

Fucoidan oligosaccharides possesses diverse physicochemical and biological activities. Specific glycoside hydrolases are valuable tools for degrading polysaccharides to produce oligosaccharides. In this study, BcFucA, a novel fucosidase belonging to GH95 family from Bacillus cereus 2–8, was cloned into pET21a vector, expressed in E. coli BL21 (DE3) and characterized. The protein consists of 1136 amino acid residues encoded by 3411 bases and has a molecular weight of 125.35 kDa. The optimal temperature and pH of this enzyme are 50 °C and pH 4.0. In addition, this study showed that the unknown function domain (encoding Lys261-Thr681) defined as a linker is quite important for its activity. The obtained novel enzyme BcFucA will contribute to the effective degradation of fucoidan and future industrial applications.

Design of a novel multiple epitope-based vaccine: An immunoinformatics approach to combat SARS-CoV-2 strains

BackgroundSince the SARS-CoV-2 outbreak in December 2019 in Wuhan, China, the virus has infected more than 153 million individuals across the world due to its human-to-human transmission. The USA is the most affected country having more than 32-million cases till date. Sudden high fever, pneumonia and organ failure have been observed in infected individuals. ObjectivesIn the current situation of emerging viral disease, there is no specific vaccine, or any therapeutics available for SARS-CoV-2, thus there is a dire need to design a potential vaccine to combat the virus by developing immunity in the population. The purpose of present study was to develop a potential vaccine by targeting B and T-cell epitopes using bioinformatics approaches. MethodsB- and T-cell epitopes are predicted from novel M protein-SARS-CoV-2 for the development of a unique multiple epitope vaccine by applying bioinformatics approaches. These epitopes were analyzed and selected for their immunogenicity, antigenicity scores, and toxicity in correspondence to their ability to trigger immune response. In combination to epitopes, best multi-epitope of potential immunogenic property was constructed. The epitopes were joined using EAAAK, AAY and GPGPG linkers. ResultsThe constructed vaccine showed good results of worldwide population coverage and promising immune response. This constructed vaccine was subjected to in-silico immune simulations by C-ImmSim. Chimeric protein construct was cloned into PET28a (+) vector for expression study in Escherichia coli using snapgene. ConclusionThis vaccine design proved effective in various computer-based immune response analysis as well as showed good population coverage. This study is solely dependent on developing M protein-based vaccine, and these in silico findings would be a breakthrough in the development of an effective vaccine to eradicate SARS-CoV-2 globally.

Cloning,expression and immunological identification of <italic>Der f</italic> 35 allergen from <italic>Dermatophagoides farinae</italic>

Dermatophagoides farinae is an important allergen that induces allergic diseases and type I allergic reaction. The identification of its immunogenicity is the basis for the study of allergic rhinitis and other allergic diseases. First, primers were designed according to the Der f 35 gene sequence (GenBank: LC175222.1), and the total RNA was extracted from dust mite, use it as a template for Der f 35 reverse transcription polymerase chain reaction amplification. Then, the pET-32a vector was ligated with the Der f 35 gene and transfer it into Escherichia coli (E.coli) TOP 10 cloned bacteria, and 1 mL of the cloning bacteria liquid was aspirated for sequencing. The high purity plasmid was extracted and transferred to Rosetta (DE3) cells. Expression of the objective protein Der f 35 was induced, purified and immunologically identified. Homology comparison analysis, phylogenetic tree construction and secondary structure prediction was then performed. Results showed that the fragment of the Der f 35 gene was about 436 base pair (bp). The molecular weight of the recombinant protein Der f 35 was 30 ku (1 u =1 D), which was consistent with the expected results. The results of bioinformatics evolutionary tree showed that the dust mite was close to house dust mite, blomia tropicalis, scaly mite, sheep itchy mite and ear free mite. Secondary structure prediction indicated that Der f 35 amino acid sequence consists of two α helices, six β folds, two β turns and ten random coil fragments. Der f 35 gene was successfully cloned, and the coded protein was expressed and purified in E. coli with good reactivity. It lays a foundation for the study of allergic diseases of dust mites.

Combination of highly antigenic nucleoproteins to inaugurate a cross-reactive next generation vaccine candidate against Arenaviridae family

Arenaviral infections often result lethal hemorrhagic fevers, affecting primarily in African and South American regions. To date, there is no FDA-approved licensed vaccine against arenaviruses and treatments have been limited to supportive therapy. Hence, the study was employed to design a highly immunogenic cross-reactive vaccine against Arenaviridae family using reverse vaccinology approach. The whole proteome of Lassa virus (LASV), Lymphocytic Choriomeningitis virus (LCMV), Lujo virus and Guanarito virus were retrieved and assessed to determine the most antigenic viral proteins. Both T-cell and B-cell epitopes were predicted and screened based on transmembrane topology, antigenicity, allergenicity, toxicity and molecular docking analysis. The final constructs were designed using different adjuvants, top epitopes, PADRE sequence and respective linkers and were assessed for the efficacy, safety, stability and molecular cloning purposes. The proposed epitopes were highly conserved (84%–100%) and showed greater cumulative population coverage. Moreover, T cell epitope GWPYIGSRS was conserved in Junin virus (Argentine mammarenavirus) and Sabia virus (Brazilian mammarenavirus), while B cell epitope NLLYKICLSG was conserved in Machupo virus (Bolivian mammarenavirus) and Sabia virus, indicating the possibility of final vaccine construct to confer a broad range immunity in the host. Docking analysis of the refined vaccine with different MHC molecules and human immune receptors were biologically significant. The vaccine-receptor (V1-TLR3) complex showed minimal deformability at molecular level and was compatible for cloning into pET28a(+) vector of E. coli strain K12. The study could be helpful in developing vaccine to combat arenaviral infections in the future. However, further in vitro and in vivo trials using model animals are highly recommended for the experimental validation of our findings.

Heterologous expression of a novel serine palmitoyltransferase from Sphingobium chungbukense

In the described experiments, we identify and clone a novel serine palmitoyltransferase (SPT) isolated from the gram-negative obligatory aerobic bacteria Sphingobium chungbukense strain KCTC2955. SPT catalyzes the first rate-limiting reaction in the de novo biosynthesis of sphingolipids, in S. chungbukense. The DNA sequence of the cloned SPT gene (1791 bp) has an open reading frame encoding 405 amino acids with a predicted molecular mass of 41 kDa; the sequence contains the consensus PLP-binding motif (GTFSKSXXXXGG) which is conserved among serine palmitoyltransferase. The ribosomal binding site GGGA lays 6 bp upstream of the ORF of 1218 bp, which is initiated by an ATG codon and terminated by TGA. The cloned SPT gene succeeded in overproducing the SPT protein under the T7 promoter of the expression vector pET 21a in Escherichia coli BL21(DE3), in which the product amounted to about 20–30% of the total protein isolated from the cell extract. Sphingobium SPT showed about a 68.4–89.3% homology with other enzymes in the serine palmitoyltransferase family, and amino acid residues proposed to be involved in catalysis were conserved. The activity of purified recombinant-SPT using Ni-NTA metal-affinity chromatography was measured via the radioactivity of the [3H]-3-ketodihydrosphingosone (KDS) formation using l-[3H]-serine. We have identified a novel SPT gene from S. chungbukense KCTC2955 and overexpressed the recombinant SPT in E. coli. This study is a first step in understanding the reaction mechanism of SPT in bacteria as well as the first focused on the genus Sphingobium.

Functional characterization of Helianthus annuus phytochelatin synthase (HaPCS): Gene expression and protein profiles of HaPCS responding to arsenic and evaluation of arsenic accumulation in engineered bacteria expressing HaPCS

Arsenic is the most prevalent environmental toxic element and causes a variety health effects. Phytochelatins (PCs) are naturally occurring peptides with high affinity toward metal(loid)s that are present in plants and fungi. Here, the gene encoding phytochelatin synthase from Helianthus annuus (HaPCS) was isolated and cloned in expression vectors pET28a and pET41a. The resulting constructs were transformed into Escherichia coli strains Rosetta (DE3) and Rosetta gami 2. Following the induction with Isopropyl β-D-1-thiogalactopyranoside, the enzyme GST-HaPCS was expressed in the strains Rg-41a-HaPCS and R-41a-HaPCS and the enzyme His-HaPCS was expressed in R-28a-HaPCS. The comparison of soluble and insoluble extracted fractions revealed that the expression of GST-HaPCS in the strain Rg-41a-HaPCS is the optimum method for expression of this enzyme. The recombinant and pure GST-HaPCS catalysed PCs in vitro only in the presence of Cd2+, As3+ and As5+. The presence of Cd2+ activated the enzyme GST-HaPCS more than As3+/As5+. Therefore the enzyme GST-HaPCS seems to be differentially activated by different metal(loid)s. The strain Rg-41a-PCS was able to synthesise PCs when the medium was supplemented by As3+, As5+ and Cd2+. In these conditions the new strains were able to accumulate 10.67 μmol/g DCW As5+, 7.58 μmol/g DCW As3+ and 25.97 and 25.97 μmol/g DCW in medium containing 0.5 mM of each of these metal(loid)s. Furthermore in the present work we found that there was no difference between the expressions of HaPCS in H. annuus in response to different metal(loid)s. Therefore the higher accumulation of PCs and subsequently higher accumulation of Cd2+ could be due to the higher activity of HaPCS in the presence of this metal ion. This study introduces a good candidate gene from plant source with high ability for synthesis of PCs and accumulation of arsenic and cadmium.

Identification and Heterologous Production of a Lipase from Geobacillus kaustophilus DSM 7263T and Tailoring Its N-Terminal by a His-Tag Epitope

Lipases are versatile biocatalysts with many biotechnological applications and the necessity of screening, production and characterization of new lipases from diverse microbial strains to meet industrial needs is constantly emerging. In this study, the lipase gene (gklip) from a thermophilic bacterium, Geobacillus kaustophilus DSM 7263T was cloned into the pET28a ( +) vector with N-terminal 6xHis-tag. The recombinant gklip gene was heterologously expressed in host E. coli BL21 (DE3) cells and purified by Ni-NTA affinity chromatography. Histidine tag was removed from the purified 6xHistag-Gklip enzyme with thrombin enzyme and the molecular mass was determined to be approximately 43kDa by SDS-PAGE. Gklip showed optimal activity at pH 8.0 and 50°C. The specific hydrolytic activities against substrates were significantly increased by the removal of the His-tag. Km and kcat values of Gklip against p-nitrophenyl palmitate (pNPP, 4-nitrophenyl palmitate) as the target substrate were found to be as 1.22mM and 417.1min-1, respectively. Removing His-tag changed the substrate preference of the enzyme leading to maximum lipolytic activity towards C10 and C12 lipids. Similarly, the activity against coconut oil that containing 62% medium-chain fatty acids was significantly higher than other oils. Furthermore, preservation of activity in the presence of inhibitors, organic solvents support the effect of lid structure of the enzyme.

Polymorphic membrane protein 20G: A promising diagnostic biomarker for specific detection of Chlamydia psittaci infection

Psittacosis is a zoonotic disease caused by Chlamydia psittaci (C. psittaci), leading to high risk for animal industry and human health. Lack of reliable commercial kits and effective vaccines is hampering control of C. psittaci infection. Polymorphic outer membrane protein Gs (PmpGs) are enriched in diverse C. psittaci, and its role are unclear during C. psittaci infection. In the present study, pmp20G gene was cloned into pET-28a vector and then the constructed plasmid was transferred into Escherichia coli Rossetta (DE3). After denaturation and renaturation, the recombinant Pmp20G-N was identified by SDS-PAGE and Western blot. Afterwards Pmp20G-N was used as the coating antigen to develop an indirect ELISA (I-ELISA) assay. Both the specificity and sensitivity of Pmp20G-N ELISA were 100%, while the MOMP-ELISA had 93.65% sensitivity and 98.94% specificity, respectively. The concordance between MOMP-ELISA and Pmp20G-N ELISA assay was 98.1%. Hence, Pmp20G-N ELISA has the potential to be a diagnostic antigen for detection C. psittaci antibody. However, further studies are needed to be done for differentiating C. psittaci from Chlamydia spp. and other C.psittaci-specific serovars using Pmp20G-N ELISA.

Production and Characterization of Monoclonal Antibodies Against N Protein of Rift Valley Fever Virus.

The DNA fragment encoding predicted main antigenic region, aa 14-245 on N protein of Rift Valley virus (RVFV) was cloned into the vector pET-28a (+) and p3xFLAG-CMV-10. The recombinant pET-28a-N1 protein was expressed in Escherichia coli BL21 (DE3) with 1 mM isopropyl-b-thio-galactopyranoside at 37°C for 5 hours, and purified by protein purifier. Three monoclonal antibodies (mAbs) named 3A5, 3A6, and 3A7 against N protein were obtained by fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from pET-28a-N1 protein-immunized mice. Finally, the mAbs were characterized by enzyme-linked immunosorbent assays, indirect immunofluorescent assays, and Western blot. The results show that all the mAbs possess high specificity and react with both prokaryotic and eukaryotic N protein, which could provide important materials for the research on the function of N protein and the diagnostic methods of RVFV.

<p><strong>Cloning and expression of cultural filtrate proteins from novel and native strains of <em>Mycobacterium avium </em>subspecies <em>paratuberculosis </em>and their application in ELISA based sero-diagnosis of Johne's disease</strong></p>

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is endemic in livestock leading to low per animal productivity. MAP as survives pasteurization, poses a public health problem because of high exposure to animals and humans. There is an urgent need for newer diagnostic tests with high specificity and sensitivity as the current ones suffer from lower sensitivity and specificity. In present study, six Mycobacterium avium subspecies paratuberculosis (MAP)-specific culture filtrate proteins (CFPs) were produced and evaluated for sero-diagnosis of MAP infection in goat and cattle herds in India. Genes encoding for six MAP-CFPs were amplified and cloned into easy cloning vector pJET1.2/pTZ57R followed by sub-cloning into expression vector pET28a (+)/pET22b (+) containing C-terminal Histidine. Recombinant CFPs (r-CFPs) expressions were optimized in Escherichia coli (Rosetta cells) and purified using Ni-NTA affinity chromatography. In SDS-PAGE, MAP CFPs viz., 1693c, 2168c, ModD, 85C, Pep AN and Pep AC showed 22, 24, 55, 38, 20 and 25 kDa molecular masses, respectively. Identity of these r-CFPs was further confirmed by immuno-blotting. We developed six different ELISAs using the six individual r-CFPs and one additional ELISA i.e. cocktail ELISA (c-ELISA) was prepared using cocktail of all 6 r-CFPs. The performance of all seven ELISAs were further evaluated against whole cell protoplasmic based indigenous ELISA (i-ELISA). c-ELISA showed almost similar sensitivity as shown by i-ELISA. However, individual r-CFP based ELISA could not reach up to the sensitivity of cocktail of six r-CFPs. None of the r-CFP showed any false positive (as compare to i-ELISA) thereby specificity was 100%. Results of ELISA tests based on cocktail of r-CFPs, ModD and 85C were quite similar to i-ELISA from goat sera whereas in cattle serum c-ELISA was comparable with i-ELISA. Our study showed a comparable specificity of c-ELISA for the diagnosis of JD and it may have applicability in region where disease is endemic. Future validation of c-ELISA against gold standard or confirmatory tests would give a better insight on its diagnostic potential over i-ELISA.

Characterization of pyruvate dehydrogenase complex E1 alpha and beta subunits of Mycoplasma synoviae

Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.