Prokaryotic Expression System Research Articles

In Vitro Evaluation of Immunogenicity of Recombinant OMP25 Protein Obtained from Endemic Brucella abortus Biovar 3 as Vaccine Candidate Molecule Against Animal Brucellosis.

Brucellosis is a zoonotic disease that causes serious economic losses due to factors, such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches. The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus, and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response. The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the prokaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively. It was determined that doses of the recombinant Omp25 protein greater than 0.1 μg/mL are toxic to RAW cells. Doses of 1 μg/mL and lower significantly increased inflammation due to Nitric Oxide (NO) levels. ELISA results showed that IFN-γ was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 μg/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose. Studies on cell viability and Th1-related cytokine release suggest Omp25 protein to be a promising candidate molecule for vaccine development.

Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

Molecular Characterizations and Functional Analyses of LmR2D2 in the Locusta migratoria siRNA Pathway.

Simple SummaryLocusta migratoria is a serious agricultural pest all over the world, which devastates agriculture and challenges food security and livelihoods. Recently, RNA interference (RNAi) technology has emerged as a novel strategy for managing insect pests. However, different insect species show a significant variation in RNAi efficiency. Therefore, it is necessary to better understand the RNAi mechanism. In this study, we identified an LmR2D2 gene in the L. migratoria transcriptome. After the expression of LmR2D2 was suppressed by RNAi, we found a significantly diminished RNAi efficiency against a marker gene in L. migratoria. Our binding experiments further demonstrated that the LmR2D2 protein can bind double-stranded RNA (dsRNA) in vitro. On the basis of these results, we conclude that LmR2D2 is involved in the L. migratoria siRNA pathway.Small interfering RNAs (siRNAs) are non-coding RNAs with a length of 21~23 nucleotides (nt) and present in almost all eukaryotes. The formation of siRNA is a highly conserved post-transcriptional gene-silencing mechanism mediated by key proteins, including Dicer2, Argonaute2 (Ago2) and R2D2. R2D2 has been identified as a double-stranded RNA (dsRNA)-binding protein and reported as an integral component of the siRNA pathway in Drosophila. However, the involvement of R2D2 in the siRNA pathway of Locusta migratoria is still unknown. In the present study, we identified an LmR2D2 gene from the transcriptome of L. migratoria. It consists of a 954-bp open reading frame that encodes a protein of 318 amino acid residues. Further sequence analysis revealed that LmR2D2 possesses two tandem dsRNA-binding domains (dsRBD) at the N-terminus. Analysis of the developmental expression profile of LmR2D2 indicated that its transcript level was stable in third-instar nymphs of L. migratoria, whereas the tissue-dependent expression profile exhibited high levels of expression of LmR2D2 in the testis and ovary. When LmR2D2 was silenced by RNAi, the RNAi efficiency against Lmβ-tubulin as a marker gene was significantly diminished, as indicated by the 37.7% increased Lmβ-tubulin transcript level. Additionally, the prokaryotic expression system was used to obtain the LmR2D2 supernatant protein. By incubating the LmR2D2 protein with biotin-dsRNA, we found that LmR2D2 can bind to dsRNA in vitro, which supports our conclusion that LmR2D2 plays an essential role in the siRNA pathway of L. migratoria.

Novel nucleic acid aptamer gold (Au)-nanoparticles (AuNPs-AptHLA-G5-1 and AuNPs-AptHLA-G5-2) to detect the soluble human leukocyte antigen G5 subtype (HLA-G5) in liquid samples.

BackgroundThe human leukocyte antigen G5 subtype (HLA-G5) is a major histocompatibility complex (MHC) molecule that is selectively expressed at the maternal-foetal tissue interface and is required for the successful implantation of the in vitro fertilized embryo. It is critical to detect HLA-G5, especially HLA-G5 expression in embryo fluid, during in vitro embryo incubation and culture. However, the specificity and sensitivity of traditional ELISA methods to detect sHLA-G5 are insufficient. This work aimed to explore novel nucleic acid aptamer gold (Au)-nanoparticles to detect soluble HLA-G5 in liquid samples.MethodsSoluble HLA-G5 was obtained using a prokaryotic expression system, and two novel aptamers (HLA-G5-Apt1 and HLA-G5-Apt2) detecting HLA-G5 were screened by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. Small (10 nm) gold nanoparticles (AuNPs) were incubated with AptHLAs to form two novel nucleic acid aptamers: Au-nanoparticles (AuNPs-AptHLA-G5-1 and AuNPs-AptHLA-G5-2).ResultsThe results showed that AptHLA-G5-1 and AptHLA-G5-2 have a high affinity for HLA-G5 and can detect its presence in liquid samples. Using the colorimetric sensing method, AuNPs-AptHLA-G1 had a detection limit as low as 20 ng/mL (recovery range between 98.7% to 102.0%), while AuNPs-AptHLA-G2 had a detection limit as low as 20 ng/mL (recovery range between 98.9% to 103.6%).ConclusionsOur work demonstrates that novel AuNPs are efficient detectors for HLA-G5 and are useful for diagnosis and treatment in the field of obstetrics-gynaecology.

A Method for Rapid Screening, Expression, and Purification of Antimicrobial Peptides.

In this study, a method for the rapid screening, expression and purification of antimicrobial peptides (AMPs) was developed. AMP genes were fused to a heat-resistant CL7 tag using the SLOPE method, and cloned into Escherichia coli and Pichia pastoris expression vectors. Twenty E. coli and ten P. pastoris expression vectors were constructed. Expression supernatants were heated, heteroproteins were removed, and fusion proteins were purified by nickel affinity (Ni-NTA) chromatography. Fusion proteins were digested on the column using human rhinovirus (HRV) 3C protease, and AMPs were released and further purified. Five AMPs (1, 2, 6, 13, 16) were purified using the E. coli expression system, and one AMP (13) was purified using the P. pastoris expression system. Inhibition zone and minimum inhibitory concentration (MIC) tests confirmed that one P. pastoris¬-derived and two E. coli-derived AMPs have the inhibition activity. The MIC of AMP 13 and 16 from E. coli was 24.2 μM, and the MIC of AMP 13 from P. pastoris was 8.1 μM. The combination of prokaryotic and eukaryotic expression systems expands the universality of the developed method, facilitating screening of a large number of biologically active AMPs, establishing an AMP library, and producing AMPs by industrialised biological methods.

Identification of novel B-cell epitopes on the capsid protein of type 1 porcine astrovirus, using monoclonal antibodies

Porcine astrovirus (PAstV) is prevalent in pigs worldwide and could cause clinical symptoms such as diarrhea and encephalitis. The capsid protein (Cap) of PAstV plays a determinant role for virus immunological characteristics. In this study, the major antigenic regions of PAstV1 Cap were expressed through prokaryotic expression systems and immunized to BALB/c mice. Finally, two anti-Cap monoclonal antibodies (named mAb F4-4 and D3F10) were screened by indirect immune-fluorescence assay (IFA). A series of truncated GST-fused or artificially synthesized peptides were used to detect their reactivity with the mAbs and PAstV positive serum. Two novel B cell epitopes (120-GNNTFG-125, 485-RISDPTWFSA-494) were identified by using these two mAbs. Moreover, sequence alignment result showed that epitope 120-GNNTFG-125 was highly conserved in type 1 PAstV capsid protein. Cross-reactivity analysis further confirmed the genotype-specificity of mAb F4-4. The results of this study demonstrated to be the first description of monoclonal antibody preparation and B-cell epitope mapping on PAstV capsid protein, which may provide new information on the biological significance of PAstV capsid protein and lay a foundation for the development of PAstV immunological tests and genotype diagnostic methods.

Effect of active immunization with recombinant-derived goose INH-α, AMH, and PRL fusion protein on broodiness onset and egg production in geese (Anser cygnoides)

This study was conducted to investigate the potential effects of active immunization against recombinant-derived goose inhibin-α (INH-α), anti-Müllerian hormone (AMH), and prolactin (PRL) fusion protein on broodiness onset and egg production in geese. The purified fusion proteins (INH-α, AMH, and PRL) were prepared using a prokaryotic expression system. Female Zhedong geese (10 mo old) were randomly assigned to one of 4 treatments and raised in separate pens. The geese were actively immunized with the recombinant goose INH-α, AMH, or PRL, respectively, and phosphate-buffered saline as control. The results showed the corresponding antibodies were produced when the geese were immune INH-α, AMH-, and PRL-recombinant proteins. The significantly higher luteinizing hormone contents were observed in the INH-α, AMH, and PRL recombinant protein-immunized geese, while the lower AMH hormone content only in PRL-immunized birds. AMH recombinant protein immunized geese had more large yellow follicles of ovary, while the INHα-treated birds with more other follicles compared with control geese. In addition, the geese receiving INH-α recombinant protein, the broodiness onset was about 6 d, which significantly shorter than did PBS immunization (16 d). The INHα- and PRL-immunization also resulted in 12.5 and 8.5 d shorter broody duration intervals compared to the control birds. Moreover, the lower new broodiness rate was observed in three recombinant proteins treated birds. Finally, the PRL recombinant protein-immunization resulted in an average increase of 1.34 eggs during a 40-d observation. Collectively, the data demonstrated that active immunization against recombinant proteins INH-α or AMH could promote LH hormone secretion, regulate follicle development and decrease the broodiness rate. Also, active immunization with a recombinant-derived goose PRL protein might improve egg laying performance.

Development of a subunit vaccine based on fiber2 and hexon against fowl adenovirus serotype 4

Hepatitis-hydropericardium syndrome (HHS) is widespread in China and causes high chicken mortality that results in great economic losses. A safe and effective vaccine is needed, and a subunit vaccine has potential for development. In this study, a truncated region of the FAdV-4 fiber 2 fused with coding sequence of one epitope of hexon was expressed in a prokaryotic expression system, and the immune protective effects of different doses of recombinant fiber 2 subunit vaccine on SPF chickens were compared. The recombinant fiber2 (Gly275- Pro479 aa)-hexon (Met21-Val51 aa) protein (rFH) obtained in Escherichia coli showed good solubility. The chicken survival rate at the lowest dose (2.5 μg/bird) was 75% (6/8), and at higher doses (≥5 μg/bird) was 100% (8/8) in challenge experiment. Two chickens in the 2.5 μg/bird treatment showed severe lesions, while birds in the higher dose treatments showed no obvious tissue damage as determined by histopathologic analysis of liver and spleen. Absolute quantitative real-time PCR showed no viral load in the ≥5 μg/bird treatments, but two chickens in the 2.5 μg/bird treatment had high viral loads. The challenge experience demonstrated that the rFH vaccine provided 100% protection at ≥5 μg/bird. These results suggested that rFH protein as an effective vaccine to protect against FAdV-4 and provided a new idea for the development of vaccine against HHS.

Inhibition of human lung cancer cells by anti-p21Ras scFv mediated by the activatable cell-penetrating peptide.

Activatable cell-penetrating peptide (ACPP) is a tumour-targeting cell-penetrating peptide. Here, we used ACPP to carry anti-p21Ras scFv for Ras-driven cancer therapy. The ACPP-p21Ras scFv fusion protein was prepared by a prokaryotic expression system and Ni-NTA column purification. The human tumour cell lines A549, SW480, U251 and Huh7 and the normal cell line BEAS 2B were used to study the tumor-targeting and membrane-penetrating ability of ACPP-p21Ras scFv. The antitumour activity of ACPP-p21Ras scFv on A549 cells and H1299 cells in vitro was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, scratch wound healing, plate cloning and apoptosis assays. The penetration pathway of ACPP was determined by enhanced green fluorescent protein. The ACPP-p21Ras scFv fusion protein was successfully obtained at a concentration of 1.8 mg/ml. We found that ACPP-p21Ras scFv could penetrate tumour cell membranes with high expression of matrix metalloproteinase-2 (MMP-2), effectively inhibit the migration and proliferation of A549 cells and H1299 cells, and promote the apoptosis of A549 cells and H1299 cells. The membrane penetration experiment demonstrated that ACPP could enter A549 cells by direct penetration. The ability of ACPP to penetrate the membrane was affected by the addition of a membrane affinity inhibitor and a change in the potential difference across the cell membrane but not by the addition of endocytosis inhibitors and a change in temperature. The ACPP-p21Ras scFv fusion protein can penetrate tumour cells with MMP-2 expression and has antitumour activity against A549 cells and H1299 cells in vitro. This molecule is expected to become a potential antitumour drug for Ras gene-driven lung cancer.

Bombyx mori Apolipophorin-III inhibits Beauveria bassiana directly and through regulating expression of genes relevant to immune signaling pathways

Insect Apolipophorin-III is a multifunctional protein and also plays an important role in insect innate immunity. Early transcriptome and proteome studies indicated that the gene expression level of Bombyx mori Apolipophorin-III (BmApoLp-III) in silkworm larvae infected with Beauveria bassiana was significantly up-regulated. In this study, BmApoLp-III gene was cloned, its expression patterns in different larval tissues investigated, the BmApoLp-III protein was successfully expressed with prokaryotic expression system and its antifungal effect was verified. The results showed that the BmApoLp-III gene was expressed in all the tested tissues of the 5th instar larvae infected by B. bassiana, with the highest expression in fat body. The fungistatic zone test showed that the recombinant BmApoLp-III has a significant antifungal effect on B. bassiana. Injecting purified BmApoLp-III to the larvae delayed the onset and death of the infected larvae. Conversely, silencing BmApoLp-III gene by RNAi resulted in early morbidity and death of the infected larvae. At the same time, injecting BmApoLp-III up-regulated the expression of genes including BmβGRP4 and BmMyd88 in the Toll signaling pathway, BmCTL5 and BmHOP in the Jak/STAT signaling pathway, serine proteinase inhibitor BmSerpin5, and antimicrobial peptide BmCecA, but down-regulated the expression of BmTak1 of Imd signaling pathway; while silencing BmApoLp-III gene down-regulated the expression of BmβGRP1 and BmSpaetzle, BmCTL5 and BmHOP, BmSerpin2 and BmSerpin5, BmBAEE and BmPPO2 of relevant pathways and BmCecA, but up-regulated the expression of BmPGRP-Lc and BmTak1 of Imd pathway. These results indicate that the BmApoLp-III could not only directly inhibit B. bassiana, but also participate in regulation of the expression of immune signaling pathway related genes, promote the expression of immune effectors, and indirectly inhibit the reproduction of B. bassiana in the silkworm.

Enzymatic characteristics and preventive effect of leucine aminopeptidase against Echinococcus multilocularis

Alveolar echinococcosis, a parasitic zoonotic disease caused by the larval stage of Echinococcus multilocularis infection, is a global epidemic in Eurasia and North America. Leucine aminopeptidase (LAP) of the M17 peptidase family could act on an ideal target antigen in diagnosis and prevention of parasitic diseases (schistosomiasis, malaria, fascioliasis) because of its good immunogenicity. In this study, the bioinformatic and enzymatic characterizations of recombinant Echinococcus multilocularis LAP (rEm-LAP) were evaluated. A prokaryotic expression system for rEm-LAP protein was established and its immunogenicity and preventive efficacy were demonstrated in a BALB/c mice model. This is the first report about the LAP of Echinococcus multilocularis and with a 57.4 KD purified rEm-LAP protein successfully expressed by pCzn1-LAP in Escherichia coli BL-21 cells. Enzymatic analysis results showed optimal rEm-LAP activity at pH 9. Serum indirect ELISA demonstrated that rEm-LAP could induce a Th1 and Th2 mixed-type immunological response and produce high levels of IgG, IgG1, IgG2a, IgM, and IgA. Furthermore, serum IFN-γ and IL-4 secretion were increased compared with the control groups. Finally, vaccination with rEm-LAP significantly decreased both the number and size of the cysts in Echinococcus multilocularis metacestode infected mice model. The current study provides evidence that rEm-LAP could be a potential vaccine antigen of Echinococcus multilocularis.

Epitope screening of the major capsid protein within grouper iridovirus of Taiwan and the immunoprotective effect with SWCNTs as the vaccine carrier

Iridovirus can cause a mass of death in grouper, leading to huge economic loss in recent years. At present, practical vaccine is still the best way to control the outbreak of this virus. Many researches had indicated that the major capsid protein (MCP) of grouper iridovirus of Taiwan (TGIV) is an effective antigen to induce a specific immune response in grouper. However, these traditional vaccines that based on large proteins or whole organisms are faced with challenges because of the unnecessary antigenic load. Thus, in this study, we screened the dominant linear epitope within the MCP of TGIV and then, a new peptide vaccine (P2) was developed via prokaryotic expression system. Furthermore, SWCNTs was used as a vaccine carrier to enhance the immunoprotective effect. To evaluate the immunoprotective effect of this vaccine, a total of 245 fish were vaccinated with P2 (5, 10, 20 mg L−1) and SWCNTs-P2 (5, 10, 20 mg L−1) via immersion before being challenged with live TGIV at 28 days post immunization (d.p.i.). Results showed that the serum antibody titer, enzymatic activity, expression level of some immune-related genes (CC chemokine, IgM and TNF-α) and survival rate were significantly increased (SWCNTs-P2, 20 mg L−1, 100%) compared to the control group (0%). These results indicated that this peptide vaccine could effectively induce specific immune response in vaccinated groupers. Functionalized SWCNTs could serve as a carrier of the peptide vaccine to enhance the immunoprotective effect via immersion. To sum up, epitope screening might be a potential way to develop an effective vaccine nowadays, and SWCNTs might provide a practical method that can be used in large-scale vaccination, especially for juvenile fish, to fight against diseases in aquaculture industry.

The construction of recombinant Lactobacillus casei expressing hemagglutinin-neuraminidase protein and its immune response in chickens

Newcastle disease virus (NDV) is one of the most important diseases in poultry. The present study generated recombinant surface-displayed Lactobacillus casei (L. casei) expressing the hemagglutinin-neuraminidase (HN) of NDV (Lc-pPG-HN) and a live pPG vector (Lc-pPG) and evaluated their immunogenicity. A 1670 bp HN gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in the resulting recombinant Lc-pPG-HN (surface displayed) strain was verified using Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, and the molecular weight of the corresponding protein was 63 kDa. A fluorescent signal for Lc-pPG-HN was observed using fluorescence microscopy. A total of 270 healthy chicks were divided into three treatment groups. Five replicates were used for each treatment, while six chicks were used per replicate. The following three treatment groups were used: physiological saline group (Control), Lc-pPG group and recombinant vaccine group (Lc-pPG-HN). The primary immunization and booster immunization of the chicks were performed via oral administration on 1 and 10 days old. Tissue and blood samples were collected from chickens that received oral recombinant L. casei strains on 1, 7, 14 and 21 days post-immunization for immune-related index analyses. Chickens orally immunized with Lc-pPG-HN showed significantly increased body weights and immune organ indices. Oral immunization with Lc-pPG-HN also enhanced the concentrations of serum interleukin-2 (IL-2), interferon-γ (IFN-γ), intestinal lavage fluid secretory immunoglobulin A (SIgA) and histomorphological development of the small intestine. Our results also indicated that recombinant L. casei significantly increased Lactobacillus and Bifidobacterium colonization and decreased the relative abundance of Escherichia coli (E. coli) in the chicken caecum. Similar enhancement effects from hemagglutination inhibition were also observed in the antibody titers. Oral administration of Lc-pPG-HN effectively protected against NDV and alleviated the symptoms of the NDV challenge. In summary, recombinant L. casei had positive impacts on the performance, immunological function, gut development, and microbiota of growing chicks and may be a potential therapeutic candidate against NDV.

In Silico designing and immunogenic production of the multimeric CfaB*ST, CfaE, LTB antigen as a peptide vaccine against Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the most frequent bacterial cause of diarrhea particularly reported in children of developing countries and also travelers. Enterotoxins and colonization factor antigens (CFAs) are two major virulence factors in ETEC pathogenesis. Colonization factor antigen I (CFA/I) includes major pilin subunit CfaB, and a minor adhesive subunit (CfaE), and enterotoxins consisting of heat-labile toxin subunit B (LTB) and heat-stable toxin (ST). Chimeric proteins (CCL) carrying epitopes and adjuvant sequences increase the possibility of eliciting a broad cellular or effective immune response. In the present study, a chimeric candidate vaccine containing CfaB*ST, CfaE, and LTB (CCL) was designed via in silico techniques. This chimeric gene was synthesized by using codon usage of E. coli for increasing the expression of the recombinant protein. After designing the chimeric construct, it showed a high antigenicity index estimated by the vaxiJen server. Linear and conformational B-cell epitopes were identified and indicated suitable immunogenicity of this multimeric recombinant protein. Thermodynamic analyses for mRNA structures revealed the appropriate folding of the RNA representative good stability of this molecule. In silico scanning was done to predict the 3D structure of the protein, and modeling was validated using the Ramachandran plot analysis. The chimeric protein (rCCL) was expressed in a prokaryotic expression system (E. coli), purified, and analyzed for their immunogenic properties. It was revealed that the production of a high titer of antibody produced in immunized mice could neutralize the ETEC using the rabbit ileal loop tests. The results indicated that the protein inferred from the recombinant protein (rCCL) construct could act as a proper vaccine candidate against three critical causative agents of diarrheal bacteria at the same time.

VqBGH40a isolated from Chinese wild Vitis quinquangularis degrades trans-piceid and enhances trans-resveratrol

Resveratrol (3,5,4’-trihydroxy-stilbene) is a phytoalexin that can prevent plants from pathogen attacks. Piceid is the glycosylation product of resveratrol and the main storage form of stilbenes in grapevines. Here, we reported the function of a β-glycoside hydrolase gene, VqBGH40a, from the Chinese wild grapevine Vitis quinquangularis accession Danfeng-2 in the regulation of plant resistance to powdery mildew (Uncinula necator). VqBGH40a belonging to β-glycoside hydrolase family 1 encoded 506 amino acids and was located on the cytomembrane. Its optimal induction condition was 28 or 30℃, for 4 h, with 0.1 mM IPTG in a prokaryotic expression system. Enzyme activity detection showed that purified VqBGH40a could hydrolyze trans-piceid to form trans-resveratrol in vitro. VqBGH40a was transiently overexpressed in Danfeng-2 leaves and then artificially inoculated with powdery mildew showed that VqBGH40a protein could hydrolyze trans-piceid in vivo. Additionally, a comparative family analysis between VqBGH40a and 38 VviBGHs was performed. Overall, these results demonstrate that VqBGH40a can hydrolyze trans-piceid, enhance trans-resveratrol content, and participate in the defense mechanism of grapevine against powdery mildew.

Functional characteristics of a novel odorant binding protein in the legume pod borer, Maruca vitrata

Insect olfaction system plays a key role in the foraging food, pollination, mating, oviposition, reproduction and other insect physiological behavior. Odorant binding protein are widely found in the various olfactory sensilla of different insect antennae and involved in chemical signals discrimination from natural environment. In this study, a novel OBP gene, MvitOBP3 is identified from the legume pod borer, Maruca vitrata, which it mainly harms important legume vegetables including cowpea, soybean and lablab bean. Real-time PCR results demonstrated that MvitOBP3 gene was abundantly expressed in the antennal tissue of M. vitrata, while low levels were distributed in the head, thorax, abdomen, leg and wing of adult moths. The recombinant OBP3 protein was purified using the prokaryotic expression and affinity chromatography system. Fluorescence competitive binding experiments indicated that that MvitOBP3 protein exhibited greater binding affinities with host-plant flower volatiles including Butanoic acid butyl ester, Limonene, 1H-indol-4-ol and 2-methyl-3-phenylpropanal, highlighting they may have attractant activities for the oviposition of female moths on the legume vegetables. Moreover, protein homology modeling and molecular docking analysis revealed that there are six amino acid sites of MvitOBP3 involved in the binding of the host-plant volatiles. These findings will further promote to understand the key role of odorant binding protein during host perception and oviposition of M. vitrata moths, which improve the efficiency of semiochemical-based prevention and monitoring for this pest in the legume vegetables field.

An improved lentiviral system for efficient expression and purification of β-defensins in mammalian cells.

β-Defensins are a family of conserved small cationic antimicrobial peptides with different significant biological functions.The majority of mammalian β-defensins are expressed in epididymis, and many of them are predicted to have post-translational modifications. However, only a few of its members have been well studied due to the limitations of expressing and purifying bioactive proteins with correct post-translational modifications efficiently.Here we developed a novel Fc tagged lentiviral system and Fc tagged prokaryotic expression systems provided new options for β-defensins expression and purification.The novel lentiviral system contains a secretive signal peptide, an N-terminal IgG Fc tag, a green fluorescent protein (GFP), and a puromycin selection marker to facilitate efficient expression and fast purification of β-defensins by protein A magnetic or agarose beads.It also enables stable and large-scale expression of β-defensins with regular biological activities and post-translational modification.Purified β-defensins such as Bin1b and a novel human β-defensin hBD129 showed antimicrobial activity, immuno-regulatory activity, and expected post-translational phosphorylation, which were not found in Escherichia coli (E. coli) in expressed form.Furthermore, we successfully applied the novel system to identify mBin1b interacting proteins, explaining Bin1b in a better way. These results suggest that the novel lentiviral system is a powerful approach to produce correct post-translational processed β-defensins with bioactivities and is useful to identify their interacting proteins. This study has laid the foundation for future studies to characterize function and mechanism of novel β-defensins.

Functional Characterization of Olfactory Proteins Involved in Chemoreception of Galeruca daurica.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) play a fundamental role in insect olfaction. Galeruca daurica (Joannis) is a new pest with outbreak status in the Inner Mongolia grasslands, northern China. In this study, six olfactory protein genes (GdauOBP1, GdauOBP6, GdauOBP10, GdauOBP15, GdauCSP4, and GdauCSP5) were cloned by RACE and expressed by constructing a prokaryotic expression system. Their binding affinities to 13 compounds from host volatiles (Allium mongolicum) were determined by fluorescence-binding assay. In order to further explore the olfactory functions of GdauOBP15 and GdauCSP5, RNA interference (RNAi) and electroantennogram (EAG) experiments were conducted. Ligand-binding assays showed that the binding properties of the six recombinant proteins to the tested volatiles were different. GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 could bind several tested ligands of host plants. It was suspected that GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 were related to the host location in G. daurica. We also found that there were different EAG responses between males and females when the GdauOBP15 and GdauCSP5 genes were silenced by RNAi. The EAG response of G. daurica females to 2-hexenal was significantly decreased in dsRNA-OBP15-injected treatment compared to the control, and the dsRNA-CSP5-treated females significantly reduced EAG response to eight tested host volatiles (1,3-dithiane, 2-hexenal, methyl benzoate, dimethyl trisulfide, myrcene, hexanal, 1,3,5-cycloheptatriene, and p-xylene). However, the EAG response had no significant difference in males. Both GdauOBP15 and GdauCSP5 may have different functions between males and females in G. daurica and may play more important roles in females searching for host plants.

Optimized expression and purification of a soluble BMP2 variant based on in-silico design

Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of l-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.

Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant

Background: Heat shock proteins (Hsps) are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s) have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents. Objectives: The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70) as adjuvant or antigen carrier. Materials and Methods: Cloning of M. tuberculosis heat shock protein (MTB-Hsp70) amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed. Results: Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI). Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freund’s adjuvant. Conclusion: Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freund’s adjuvant. Further, studies need to be carried out to explore its applicability as carrier of antigen.