Expression Vector pET-28a Research Articles

DARPin Ec1-LMWP protein scaffold in targeted delivery of siRNA molecules through EpCAM cancer stem cell marker.

This study is to investigate the binding ability of Designed Ankyrin Repeat Proteins type Ec1that was fused to Low Molecular Weight Protamine (DARPin Ec1-LMWP) protein scaffold to the Epithelial Cell Adhesion Molecule (EpCAM) Cancer Stem Cell (CSC) marker and its efficiency in targeted delivery of small interfering RNA (siRNA) molecules into the studied cells. Gene fragment encoding the DARPIn Ec1-LMWP fusion protein was subcloned into pET28a expression vector following molecular docking studies performed to examine the affinity of the fusion protein towards EpCAM marker. The binding of the siRNA to the expressed fusion protein was tested through native PAGE. The toxicity of the fusion protein was tested by MTT assay. Attachment of the complex to the EpCAM marker was investigated by flow cytometry and delivery of siRNA into the cells was assessed by fluorescence microscopy. The expressed 21.6kDa DARPin Ec1-LMWP fusion protein was purified and showed no cytotoxicity on tested cells. Arginine rich LMWP was efficiently bounded to the negatively charged siRNA molecule. Successful attachment of the fusion protein:siRNA complex to the EpCAM marker and its internalization into MCF-7 breast cancer cell line were confirmed. Here for the first time the recombinant DARPin Ec1-LMWP protein scaffold was designed and tested for targeting EpCAM surface marker and successful internalization of the siRNA into MCF-7 cells.

Immunocytochemical Localization of the Kinetochore Protein Nuf2p on the Gametophyte Chromosomes of a Cultivar of Saccharina (Phaeophyta)

Nuf2p, a centromere-associated protein, is an indispensable component of Ndc80 complex which is important for stable microtubule-kinetochore attachment, chromosome pairing and spindle assembly in mitosis. Based upon contig sequences coding for Nuf2p from a pyrosequencing transcriptome of Saccharina japonica, primers were designed for molecular cloning of this kinetochore protein. The open reading frame (ORF) of this gene SjNuf2 was 1,452 bp long, encoding a putative protein consisting of 483 amino acids. Multi-sequence alignment showed SjNuf2p possessed several conserved domains of Nuf2, Spc7, SPT2 and MIT. To illustrate the location of SjNuf2p using immunocytochemistry, its ORF was inserted into a prokaryotic expression vector pET-28a. Then the recombinant SjNuf2p (rSjNuf2p) was induced to be expressed in Escherichia coli. The rSjNuf2p was verified by Western blotting with the commercial anti-6His-tag antibody and then purified using affinity chromatography. The polyclonal antibody of anti-SjNuf2p was raised against the purified rSjNuf2p. Western blotting with this prepared antibody showed the presence of SjNuf2p in the gametophytes of a Saccharina cultivar. SjNuf2p and alpha/beta-tubulin were subsequently co-localized to the paraffin sections of kelp chromosomes with their corresponding antibodies, thus illustrating that SjNuf2p could reside at the kelp centromeres. This study laid a solid foundation for kelp centromere and karyotype analyses.

High Titer of Antibody Against Pneumococcal IgA1 Protease in Healthy Individuals

Background and Objectives: Considering rising antibiotic resistance in various strains of Streptococcus pneumoniae, there is a need to find new immunogenic candidates for developing pneumococcal vaccines. Immunoglobulin A1 (IgA1) protease is one of the virulence factors playing an important role in the pathogenesis of S. pneumoniae infections. In the present study, we aimed to evaluate the titer of antibody against pneumococcal recombinant IgA1 protease in the serum of healthy humans. Materials and Methods: A part of the IgA1 protease gene (705 bp) from S. pneumonia ATCC 49619 was amplified by PCR and then digested using restriction enzymes and ligated by the pET28a expression vector. The recombinant protein was expressed in E. coli BL21 strain. Affinity chromatography was used to purify the protein. The titer of antibody against the recombinant protease was determined in healthy individuals in three age groups of <2, 2-40, and > 40 years using indirect Enzyme-Linked Immunosorbent Assay (ELISA). Results: The expression and purification of the IgA1 recombinant protease were successful. The concentration of the purified protein was determined as 1.013 mg/ml using the NanoDrop method. The titer of anti-recombinant IgA1 protease antibody (20, 40, 80 and 160) showed a significant correlation with age (p-value<0.05). According to our results, the antibody titer was desirable, especially in individuals over two years old. Conclusion: In the present study, desirable antibody titers against the pneumococcal recombinant IgA1 protease were seen in the three groups’ serum of healthy individuals. However, a significant correlation was not totally observed among groups.

Dataset on recombinant expression of an ancient chitinase gene from different species of Leishmania parasites in bacteria and in Spodoptera frugiperda cells using baculovirus.

The data presented here is related to negative results obtained with the recombinant expression of chitinase from four species of Leishmania parasites in two expression systems, performed in order to investigate the molecular characteristics of the Leishmania chitinase and its possible application in leishmaniasis diagnosis. Thus, heterologous Leishmania sp chitinase proteins were expressed in bacteria using the prokaryotic expression vector pET28a and Escherichia coli Mach-T1, and in Spodoptera frugiperda (Sf9) insect cells, using the eukaryotic bac-to-bac expression system (Thermo Fisher Scientific) to produce recombinant baculoviruses to infect Sf9. Biochemical and cellular analysis of the various recombinant forms of the Leishmania sp chitinase produced in prokaryotic and eukaryotic expression systems were performed through SDS-PAGE and Western blotting. Chitinase produced and purified from bacteria presented low yield and formed inactive aggregates. Heterologous chitinase obtained after infection of Sf9 insect cells with all the four Leishmania species recombinant baculoviruses presented high yield of insoluble proteins. Dot-blot serological tests presented inconclusive results against the recombinant Leishmania sp chitinases produced in both expression systems. The experiments described in this paper can help researchers to avoid errors when choosing a recombinant expression systems to produce Leishmania parasites proteins for biotechnological purposes.

Chromium metabolism characteristics of coexpression of ChrA and ChrT gene

ObjectiveSerratia sp. S2 is a wild strain with chromium resistance and reduction ability. Chromium(VI) metabolic-protein-coding gene ChrA and ChrT were cloned from Serratia sp. S2, and ligated with prokaryotic expression vectors pET-28a (+) and transformed into E. coli BL21 to construct ChrA, ChrT and ChrAT engineered bacteria. By studying the characteristics of Cr(VI) metabolism in engineered bacteria, the function and mechanism of the sole expression and coexpression of ChrA and ChrT genes were studied. MethodsUsing Serratia sp. S2 genome as template, ChrA and ChrT genes were amplified by PCR, and prokaryotic expression vectors was ligated to form the recombinant plasmid pET-28a (+)-ChrA, pET-28a (+)-ChrT and pET-28a (+)-ChrAT, and transformed into E. coli BL21 to construct ChrA, ChrT, ChrAT engineered bacteria. The growth curve, tolerance, and reduction of Cr(VI), the distribution of intracellular and extracellular Cr, activity of chromium reductase and intracellular oxidative stress in engineered bacteria were measured to explore the metabolic characteristics of Cr(VI) in ChrA, ChrT, ChrAT engineered bacteria. ResultsChrA, ChrT and ChrAT engineered bacteria were successfully constructed by gene recombination technology. The tolerance to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrA > ChrT > Control (P < 0.05), and the reduction ability to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrT > ChrA (P < 0.05). The chromium distribution experiments confirmed that Cr(VI) and Cr(III) were the main valence states. Effect of electron donors on chromium reductase activity was NADPH > NADH > non-NAD(P)H (P < 0.05). The activity of chromium reductase increased significantly with NAD(P)H (P < 0.05). The Glutathione and NPSH (Non-protein Sulfhydryl) levels of ChrA, ChrAT engineered bacteria increased significantly (P < 0.05) under the condition of Cr(VI), but there was no significant difference in the indexes of ChrT engineered bacteria (P > 0.05). ConclusionChrAT engineered bacteria possesses resistance and reduction abilities of Cr(VI). ChrA protein endows the strain with the ability to resist Cr(VI). ChrT protein reduces Cr(VI) to Cr(III) by using NAD(P)H as electronic donor. The reduction process promotes the production of GSH, GSSG and NPSH to maintain the intracellular reduction state, which further improves the Cr(VI) tolerance and reduction ability of ChrAT engineered bacteria.

Comparson of the immunogenicity of genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens

To screen the best genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens, the prMEIII gene, the polytope gene and the prMEIII-polytope fusion gene of the GenotypeⅠJapanese encephalitis virus GS strain were cloned into prokaryotic expression vector pET-30a. The recombinant proteins were obtained after the induction and purification. The prepared recombinant proteins were immunized to mice, and the immunogenicity of the subunit vaccine candidate antigens was evaluated through monitoring the humoral immune response by ELISA, detecting the neutralizing antibody titer by plaque reduction neutralization test, and testing the cell-mediated immune response by lymphocyte proliferation assay and cytokine profiling. The recombinant proteins with the molecular weights of 35 (prMEIII), 28 (polytope antigen) and 57 kDa (prMEIII-polytope) induced strong humoral and cellular immune responses in mice. Compared with prMEIII-polytope and polytope proteins, the prMEIII protein induced a significant expression of IL-2 and IFN-γ (P<0.05) and the significant lymphoproliferation of splenocytes (P<0.05). The neutralizing antibody titer induced by the prMEIII protein was close to that induced by the commercial attenuated vaccine SA14-14-2 (P>0.05). The study suggests that the prMEIII protein can be used for the development of the Japanese encephalitis virus subunit vaccine.

Preparation and characterization of HBc virus like particles with site-directed coupling function

Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.

Expression of the rabies virus nucleoprotein and matrix protein in a prokaryotic system at high-levels: An efficacious production method

Rabies virus (RABV) causes a zoonotic and fatal infectious disease among all warm-blooded animals including humans that continues to give a great burden on public health and the global economy especially in developing countries such as Iran. In this study, we describe a convenient and low-cost system to express the nucleoprotein (NP) and matrix protein (MP) of rabies virus. NP gene (Pasteur virus (PV) strain) and MP gene (challenge virus standard (CVS) strain) were amplified and cloned into pET28a expression vector separately. Then both genes were expressed in Escherichia coli, Rosetta strain, after induction with IPTG. Expression of the proteins was analyzed by SDS-PAGE and western blot. NP and MP were purified by Anti His Tag Column chromatography. Our results indicated the N and M genes had been successfully cloned into the expression vector and verified purification of recombinant NP and MP as 53 and 27 kDa proteins. This study showed that the NP and MP of the rabies virus can be expressed in a reproducible prokaryotic expression system using the pET28a expression vector. NP and MP can be used in vaccine production and research fields.

Heme oxygenase 1 plays a crucial role in swamp eel response to oxidative stress induced by cadmium exposure or Aeromonas hydrophila infection.

Oxidative stress contributes a lot to initiation and progression of pathological conditions. Heme oxygenase 1 (HO1), a cytoprotective enzyme, is usually upregulated to alleviate oxidative stress in vivo. The function of teleost HO1 in the response to oxidative stress induced by heavy metal exposure and in pathogenic bacterial infection remains uncertain. In the present study, both complementary DNA and genomic sequence of a HO1-like gene cloned from the liver of swamp eel (Monopterus albus) are reported. Sequence analysis showed that the putative amino acid sequence contained a conserved heme oxygenase signature and displayed higher similarity to HO1 genes of other teleosts. Expression profile of swamp eel HO1 was investigated in healthy tissues and in tissues following stimulation with pathogenic bacteria (Aeromonas hydrophila) or cadmium chloride (CdCl2) exposure. Results demonstrated that HO1 messenger RNA (mRNA) was highly expressed in the liver and relatively less in other tissues. Bacterial infection with A. hydrophila significantly changed HO1 mRNA expression in the liver, spleen, and kidney, and the mRNA expression of HO1 and Nrf2 in the liver was elevated after the fish were exposed to CdCl2. Subsequently, the swamp eel HO1 was subcloned into a pET28a expression vector and transformed into Escherichia coli BL21 (DE3). Recombinant HO1 (rHO1) was successfully induced by 0.1mmol/l IPTG and purified by Ni-NTA His Bind Resin purification system. To determine whether the rHO1 could confer stress tolerance in vitro, the viability of control and HO1-expressing E. coli under CdCl2 stress was compared by spot assay. The rHO1 protein significantly increased survival rates of the bacterial hosts. To evaluate whether intraperitoneal injection with rHO1 protected the liver of swamp eel against A. hydrophila-induced oxidative stress, mRNA expression of HO1, Nrf2, hepcidin, and IL-1β as well as the oxidative stress-related parameters (ROS and total antioxidant capacity (T-AOC)) in the liver were examined. The results showed that exogenous rHO1 could significantly upgrade the mRNA expression of HO1 and hepcidin, coupled with increased ROS and T-AOC levels. However, Nrf2 and IL-1β expression levels were significantly downregulated and upregulated, respectively. These results suggested that HO1 should not only play a protective role in oxidative stress response and its adverse effects deserved further investigation.

Production and Expression Optimization of Heterologous Serratiopeptidase

Serratiopeptidase is a bacterial metalloprotease, which is useful for the treatment of pain and inflammation. It breaks down fibrin, thins the fluids formed during inflammation and acts as an anti-biofilm agent. Because of medicinally important role of the enzyme, we aimed to study the cloning and the expression optimization of serratiopeptidase. The heat-stable serratiopeptidase (5d7w) was selected as the template. Cloning into pET28a expression vector was performed and confirmed by colony PCR and double restriction enzyme digestion. The recombinant protein was expressed in Esherichia coli BL21 and confirmed by SDS-PAGE and Western blot analysis. Different parameters such as expression vector, culture media, post-induction incubation temperature, inducer concentration, and post-induction incubation time were altered to obtain the highest amount of the recombinant protein. Serratiopeptidase was successfully cloned and expressed under optimized conditions in E. coli which confirmed by western blot analysis. The optimal conditions of expression were determined using pQE30 as vector, cultivating the host bacteria in Terrific Broth (TB) medium, at 37° C, induction by IPTG concentration equal to 0.5 mM, and cells were harvested 4 h after induction. As serratiopeptidase is a multi-potent enzyme, the expressed recombinant protein can be considered as a valuable agent for pharmaceutical applications in further studies.

Comparative Evaluation of Expression Vectors (pET32a and pET25b) in Expression of Polyepitopic Sequence Synthesized from Leishmania infantum Antigens

Comparative Evaluation of Expression Vectors (pET32a and pET25b) in Expression of Polyepitopic Sequence Synthesized from Leishmania infantum Antigens

Antitumor mechanism of MAP30 in bladder cancer T24 cells, and its potential toxic effects in mice

To investigate the antitumor mechanism of MAP30 in human bladder cell line (T24) and its potential toxic effects in mice. In this study, the biological behavior of MAP30's influence on bladder cell was investigated to reveal the antitumor mechanism and role of MAP30 in bladder cancer. MAP30 gene sequence optimized by gene synthesis codon was inserted into the prokaryotic expression vector pET-28a to produce a large amount of target protein in Escherichia coli. The protein product was obtained after purification. Membrane hydration method was used to prepare MAP30 liposome in order to enhance its membrane permeability. The effects of MAP30 on the viability, apoptosis and migration of T24 cell were assessed using 3‑(4,5‑dimethyl‑thiazol‑2‑yl)‑2,5‑diphenyl‑2H‑tetrazolium bromide (MTT), flow cytometric and TUNEL assays, respectively. Mice were transfected with bladder cancer cells for 48 h. The expressions of apoptotic and non-apoptotic proteins were determined using Western blotting. Changes in tumor volume and occurrence of metastasis were assessed using luciferase assay. After 7 days, liver and kidney were excised for histological examination. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH), and activities of catalase and glutathione peroxidase (GPx) were determined in serum or homogenate using enzyme-linked immunosorbent assay (ELISA). The yield of MAP30 after purification was significantly increased. The results of MTT assay showed that MAP30 significantly and concentration-dependently inhibited the proliferation and migration of T24 cells (p &lt; 0.05). The prepared liposomes had uniform hydrated particle size of 132.6 nm, with encapsulation efficiency of 78 %. The inhibitory effect of MAP30 liposome on T24 cells was significantly higher than that of MAP30, and MAP30 significantly increased the number of apoptotic cells (p &lt; 0.05). Western blotting showed that MAP30 significantly promoted the expression of caspase 3 (p &lt; 0.05), but did not significantly affect the expressions of bcl-2 and bax (p &gt; 0.05). It also significantly down-regulated the expressions of NF-kB, JNK and MMP2 (p &lt; 0.05). Tumor formation was significantly inhibited, and tumor volume reduced in bladder cancer-bearing mice after treatment with MAP30 (p &lt; 0.05). Histological examination showed that MAP30 induced mild histological changes in the liver and kidney of mice, and significantly increased the level of MDA at day 1 (p &lt; 0.05). It also significantly and time-dependently increased ROS, but reduced GSH levels and activities of catalase and GPx (p &lt; 0.05). However, MAP30 had no significant effect on DNA (p &gt; 0.05). The apoptotic effect of MAP30 in T24 cells is mediated via activation of caspase-3 signaling pathway. The protein produces mild histological changes in the liver and kidney of mice, but has no significant effect on DNA.

Gene Cloning, Heterologous Expression and Biochemical Characterization of A Novel Extracellular Lipase from Rhizopus Oryzae KU45.

Background: Lipases secreted from various Rhizopus oryzae strains were previously expressed in Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae and was shown to have distinct activities in response to different temperatures, metal ions, organic solvents, and specific substrates. However, until now, no other research biochemically characterized the functions of extracellular pro-lipase in a novel Rhizopus oryzae KU45.Objectives: Characterization of a novel extracellular lipase from fungus R. oryzae KU45 after heterologous expression in E. coli BL21 (DE3) strain.Materials and Methods: An extracellular lipase producing fungus was isolated from a soil sample and identified as a strain of R. oryzae by partial 18S rRNA gene sequencing. It was named as R. oryzae KU45. The lipase gene of KU45 was cloned into pET-28a expression vector and expressed in E. coli as inclusion bodies. The recombinant lipase was purified, refolded and characterized.Results: The lipase exhibited maximum activity at 45ºC, at slightly alkaline pH. It showed a broad substrate specificity acting on p-nitrophenyl esters with C8-C16 acyl groups as substrates and, many of the organic solvents and metal ions tested did not have any adverse effects on the enzyme activity.Conclusions: High stability, broad substrate specificity and activity at mesophilic temperatures in the presence of organic solvents, and metal ions make the extracellular lipase of KU45 a candidate for various biotechnological applications.

Expression and antibody preparation of recombinant truncated glycoprotein of Guertu virus

Objective To express truncated glycoprotein (Gn, Gn1, Gn2, Gn3, Gc1 and Gc2) of Guertu virus (GTV) in Escherichia coli (E.coli) cells, and prepare polyclonal antibodies against recombinant proteins Gn-His, Gc1-His and Gc2-His after purification. Methods Gene fragments encoding Gn, Gn1, Gn2, Gn3, Gc1 and Gc2 of GTV DXM strain were amplified by RT-PCR, and cloned into the prokaryotic expression vector pET-32a (+ ) to construct recombinant expression plasmids. The transformed E. coli BL21(DE3) strains carrying expression plasmids were induced by IPTG to express target proteins, which were identified by SDS-PAGE. Recombinant proteins Gn-His, Gc1-His and Gc2-His were purified by nickel affinity chromatography and detected by Western blot using GTV-positive sheep serum for analysis of their antigenicity. New Zealand white rabbits were immunized with the purified recombinant proteins. The titers and specificity of serum antibodies were analyzed by ELISA. Meanwhile, eukaryotic expression vectors pcDNA3.1-Gn, pcDNA3.1-Gc1/Gc2 were constructed and transfected into mammalian Vero cells to evaluate the binding activity of rabbit polyclonal antibodies by indirect immunofluorescence method. The specific reactivity of serum antibodies to recombinant proteins was detected by Western blot. Results Restriction enzyme analysis and DNA sequencing confirmed that the recombinant expression vectors of pET-32a-Gn, pET-32a-Gn1/Gn2/Gn3, pET-32a-Gc1/Gc2, pcDNA3.1-Gn and pcDNA3.1-Gc1/Gc2 were constructed successfully. The relative molecular mass (Mr) of the expressed recombinant proteins Gn-His, Gn1/Gn2/Gn3-His, Gc1/Gc2-His were approximately 63.4×103, 37.1×103, 31.9×103, 30.8×103, 40×103 and 54.4×103, respectively. The recombinant proteins could be recognized by GTV-positive sheep serum. The titers of polyclonal antibodies against GTV Gn, Gc1 and Gc2 were 1∶409 600, 1∶204 800 and 1∶6 400, respectively. Indirect immunofluorescence assay and Western blot showed that the prepared rabbit polyclonal antibodies could specifically react with the proteins expressed in eukaryotic cells and the recombinant proteins. Conclusions The recombinant GTV glycoproteins Gn-His and Gc1/Gc2-His were efficiently expressed and purified and characterized with good immunity. The prepared polyclonal antibodies had high titers and good specificity. This study provided reference for further studying the biological function and detection methods of GTV glycoproteins and research on vaccines. Key words: Guertu Virus; Glycoprotein; Prokaryotic expression; Eukaryotic expression; Rabbit polyclonal antibody

Bordetella pertussis’e Ait Varsayımsal Dış Zar Proteini D (BopD), Varsayımsal Dış Zar Proteini N (BopN), Lösin/ İzolösin/ Valin Bağlanma Proteini (LivJ) ve Tip 2 Fimbriya (Fim2)’nin Rekombinant Olarak İfade Edilmesi

Whooping cough (pertussis) is a contagious upper respiratory tract infection primary causative agent of which is gram-negative coccobacillus Bordetella pertussis. This disease, which may be fatal especially in infants, is characterized by repeated, violent coughing spasms caused by infection and may results in death. In this study, selected immunogenic proteins , namely Leucin/ Isoleucin/ Valin binding protein (LivJ), Fimbria type 2 (Fim2) and members of Type 3 Secretion System (TTSS) Putative Outer Protein D (BopD) and Putative Outer Protein N (BopN) expressed recombinantly . Genes of selected proteins were first cloned into pGEM®-T Easy vector and transformed to into E. coli DH5α cells, then cloned into the expression vector pET-28a(+) and transformed into E. coli BL21(DE3) cells to express the proteins. Expressed proteins were also purified by using His-tag columns.

Infectious bursal disease virus cloning and structural protein (VP2) expression in Escherichia coli

Immunosuppressive diseases like infectious bursal disease (IBD) are serious threat to the poultry meat industry. IBD virus consists of two genomic segments in which segment A encodes all structural and non-structural proteins (VP2, VP3, VP4) and segment B encodes RNA dependent RNA polymerase VP1. In IBD the host immune response is due to the VP2 capsid protein of the virus that leads to the immunosuppression of the birds. In the current research the partial VP2 clones were developed using cRNA based reversed genetic system to understand the molecular determinants of infectious bursal disease virus for pathogenic phenotype and virulence. For the expression analysis, the cloning primers were designed for amplification containing NdeI and EcoRI restriction sites for the attachment of restriction enzymes. VP2 of the IBDV was expressed in E. coli cells with the utilization of pET28a expression vector. Expression analysis of bacterial lysate on SDS-PAGE after IPTG induction of 5 hours with 1mM concentration, 30 KDa VP2 protein band was observed. This study suggests the use of recombinant VP2 (rVP2) as a subunit vaccine. Keywords: Escherichia coli; IBDV; I-ELISA; RT-PCR; rVP2 http://dx.doi.org/10.19045/bspab.2020.90080

Soluble Expression, Rapid Purification and Antiviral Activity of Recimbinant Bovine Interferon-α in Escherichia coli

In this study, the cDNA of mature bovine interferon-α (BoIFN-α) gene was cloned and expressed in the Escherichia coli expression system. Total RNA was extracted from bovine liver cells and reverse transcribed into cDNA, and then BoIFN-α gene fragment was amplified with specific primers by PCR method before being ligated into the pET-32a (+) expression vector. Since the pET-32a (+) plasmid expresses the thioredoxin A (TrxA) tag protein, it can increase the solubility of the target protein. In order to express the target protein, the pET-32a-BoIFN-α recombinant plasmid was transformed into E. coli Rosetta (DE3) strain followed by IPTG induction. It was found that the recombinant fusion protein Trx-rBoIFN-α was finally expressed in a soluble form in the host cell, accounting for about 33.45% of the total cellular protein. The protein was purified by two-step chromatography, including Ni2+-chelating Sepharose affinity chromatography and DEAE anion exchange chromatography. The purity of the purified product reached 93.0%, and the cytopathic effects (CPE) inhibition method was adopted to determine the antiviral activity of the expressed fusion protein in vesicular stomatitis virus/ Madin-Darby bovine kidney cells titration system. The specific activity of the recombinant fusion protein Trx-rBoIFN-α was measured as (3.6 ± 0.25) × 106 U/mg. After cutting of the Trx tag protein by enterokinase digestion, the remaining solo protein was confirmed as rBoIFN-α protein by Western blot assay. These findings will enable us to produce a large number of bioactive rBoIFN-α protein for future application.

Identification, recombinant expression and immunological study of Lja-SHP2 in Lampetra japonica.

The protein tyrosine phosphatase SHP2 of higher vertebrates, encoded by ptpn11 gene, catalyzes the dephosphorylation of tyrosine phosphorylation site, and plays regulatory roles in various signaling pathways by cooperating with other protein tyrosine kinase. Previous studies have shown that SHP2 plays an important role in the activation and signal transduction of T and B cells in higher vertebrates. To study the role of a SHP2 homologous molecule of lampreys (Lja-SHP2) in immune response, we cloned and expressed the open reading frame sequence of Lja-SHP2 gene in prokaryotic expression vector pET-32a. The recombinant protein was successfully expressed in E. coli and the rabbit-derived polyclonal antibody was prepared. Lampetra japonica were immunized with mixed bacteria, and the mRNA and protein of Lja-SHP2 in immune-related cells and tissues were detected by real-time quantitative PCR and Western blotting after immunization. The Lja-SHP2 mRNA and protein were not significantly affected in leukocytes and supraneural myeloid bodies, but up-regulated significantly in gill tissues (P<0.05) after challenged by mixed bacteria, which indicated that Lja-SHP2 mainly participates in the immune response of gill tissues after mixed bacteria stimulation. To further investigate whether Lja-SHP2 level was affected in three lymphocyte subsets, the B-cell mitogen lipopolysaccharide (LPS) and T-cell mitogen phytohaemagglutinin (PHA) were employed to boost the immune response in L. japonica. LPS immune stimulation increased Lja-SHP2 in leucocytes significantly compared with the control group, and but had a marginal effect on Lja-SHP2 expression in gills and supraneural myeloid bodies. PHA immune stimulation could up-regulate Lja-SHP2 level in leukocytes, gill tissues and supraneural myeloid bodies. The change of Lja-SHP2 was especially dramatical in leukocytes, which was about 2.5 times higher than that in the control group, suggesting that Lja-SHP2 is involved in the lamprey immune response mediated by PHA. Consistent with the previous finding that PHA could induce the activation of VLRA+ lymphocytes, our results showed that Lja-SHP2 might be included in the immune response of VLRA+ lymphocytes mediated by PHA in gills. This research will benefit exploring the functions of Lja-SHP2 in the immune response of lamprey and will provide clues for understanding the phylogenesis of SHP2 molecular family, and its roles in the early occurrence and evolution of adaptive immune system in higher vertebrates.

Characterization of a Mn-SOD from the desert beetle Microdera punctipennis and its increased resistance to cold stress in E. coli cells.

Insects have developed a complex network of enzymatic antioxidant systems for handling reactive oxygen species (ROS) generated during stress. Superoxide dismutases (SODs) play a determinant role in balancing ROS in insect. However, studies devoted to SODs functions in insects under cold stress are limited. In the present study, we attempted to identify and characterize a mitochondrial manganese SOD (mMn-SOD) from the desert beetle Micordera punctipennis (denoted as MpmMn-SOD) and explore its protective effects on bacteria cells under cold stress. MpmMn-SOD is composed of 202 amino acids with conserved domains required for metal ions binding and enzyme activity. RT-qPCR experiments revealed that the expression of MpmMn-SOD was ubiquitous but tissue-specific and was induced by cold stress. An E. coli (BL21) system was applied to study the function of MpmMn-SOD. The MpmMn-SOD gene was cloned into the prokaryotic expression vector pET-32a to generate a recombinant plasmid pET-32a(MpmMn-SOD). After transformation of the plasmid into E. coli BL21, the fusion protein Trx-His-MpmMn-SOD was overexpressed and identified by SDS-PAGE and Western blotting. Antioxidant activity assay showed that the death zones of the transformed bacteria BL21 (pET32a-mMn-SOD) were smaller in diameter than the control bacteria BL21 (pET32a). Survival curves under −4 °C showed that BL21 (pET32a-mMn-SOD) had significant enhanced cold resistance compared to BL21 (pET32a). Its SOD activity under −4 °C had a significant negative correlation (r = − 0.995) with superoxide anion O2•− content. Accordingly, under cold stress BL21 (pET32a-mMn-SOD) had lower electric conductivity and malondialdehyde (MDA) content than BL21 (pET32a). Taken together, our results showed that cold stress stimulated the expression of MpmMn-SOD in M. punctipennis. The E. coli cells that overexpress MpmMn-SOD increase their resistance to cold stress by scavenging ROS, and mitigate potential cell damage caused by ROS under cold conditions.

Putative Role of a Yet Uncharacterized Protein Elicitor PeBb1 Derived from Beauveria bassiana ARSEF 2860 Strain against Myzus persicae (Homoptera: Aphididae) in Brassica rapa ssp. pekinensis.

This study reports the characterization of protein elicitor PeBb1 derived from entomopathogenic fungus Beauveria bassiana ARSEF-2860 strain and its putative role in induced systemic resistance in Brassica rapa ssp. pekinensis against green peach aphid Myzus persicae. The sequence of purified elicitor protein was matched with the genomic sequence of a hypothetical protein BBA_10269 from B. bassiana ARSEF-2860 (GenBank Accession No. XP_008603588.1). The protein-encoding gene PeBb1 contained 534 bp cDNA encoding a polypeptide of 177 amino acids with a molecular mass of 19 kDa. The recombinant elicitor protein was expressed in Escherichia coli using pET-28a (+) expression vector and induced necrosis in the leaves of tobacco. The effects of elicitor protein on aphid M. persicae was determined by applying three different concentrations of PeBb1 (i.e., 26, 35, 53 μM) on B. rapa plants at 4-leaf stage and the treated plants were exposed to newly emerged (0–6 h old) apterous adult aphids. Bioassay results showed significant (p < 0.05) sub-lethal effects of the exogenous application of PeBb1 elicitor on M. persicae. Moreover, the RT-qPCR gene expression analyses showed a significant up-regulation of most of the key genes linked to ethylene (ET)- and jasmonic acid (JA)-associated plant defense pathways in elicitor-treated plants. These results not only recommend the putative utilization of PeBb1 elicitor protein in future biological pest control strategies against phloem-feeding insect pests such as M. persicae, but also help in better comprehension of the mechanisms through which beneficial fungi trigger the induced plant resistance.