scholarly journals Development of a Trivalent Construct Omp18/AhpC/FlgH Multi Epitope Peptide Vaccine Against Campylobacter jejuni

2022 ◽  
Vol 12 ◽  
Author(s):  
Hongqiang Lou ◽  
Xusheng Li ◽  
Xiusheng Sheng ◽  
Shuiqin Fang ◽  
Shaoye Wan ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Tandem Repeats ◽  
Expression System ◽  
Peptide Vaccine ◽  
Surface Protein ◽  
Infection Model ◽  
Protein Antigens ◽  
Epitope Peptide ◽  
Prokaryotic Expression System

Campylobacter jejuni (C. jejuni) is one of the major pathogens contributing to the enteritis in humans. Infection can lead to numerous complications, including but not limited to Guillain-Barre syndrome, reactive arthritis, and Reiter’s syndrome. Over the past two decades, joint efforts have been made toward developing a proper strategy of limiting the transmission of C. jejuni to humans. Nevertheless, except for biosecurity measures, no available vaccine has been developed so far. Judging from the research findings, Omp18, AhpC outer membrane protein, and FlgH flagellin subunits of C. jejuni could be adopted as surface protein antigens of C. jejuni for screening dominant epitope thanks to their strong antigenicity, expression of varying strains, and conservative sequence. In this study, bioinformatics technology was adopted to analyze the T-B antigenic epitopes of Omp18, AhpC, and FlgH in C. jejuni strain NCTC11168. Both ELISA and Western Blot methods were adopted to screen the dominant T-B combined epitope. GGS (GGCGGTAGC) sequence was adopted to connect the dominant T-B combined epitope peptides and to construct the prokaryotic expression system of tandem repeats of antigenic epitope peptides. The mouse infection model was adopted to assess the immunoprotective effect imposed by the trivalent T-B combined with antigen epitope peptide based on Omp18/AhpC/FlgH. In this study, a tandem epitope AhpC-2/Omp18-1/FlgH-1 was developed, which was composed of three epitopes and could effectively enhance the stability and antigenicity of the epitope while preserving its structure. The immunization of BALB/c mice with a tandem epitope could induce protective immunity accompanied by the generation of IgG2a antibody response through the in vitro synthesis of IFN-γ cytokines. Judging from the results of immune protection experiments, the colonization of C. jejuni declined to a significant extent, and it was expected that AhpC-2/Omp18-1/FlgH-1 could be adopted as a candidate antigen for genetic engineering vaccine of C. jejuni MAP.

scholarly journals The Plasmodium falciparum circumsporozoite protein produced in Lactococcus lactis is pure and stable

2019 ◽  
Vol 295 (2) ◽  
pp. 403-414 ◽  
Author(s):  
Susheel K. Singh ◽  
Jordan Plieskatt ◽  
Bishwanath Kumar Chourasia ◽  
Vandana Singh ◽  
Judith M. Bolscher ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Lactococcus Lactis ◽  
Malaria Vaccine ◽  
Vaccine Development ◽  
Expression System ◽  
Surface Protein ◽  
Circumsporozoite Protein ◽  
Full Length

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a pre-erythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38 NANP repeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis–derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro. We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development.

scholarly journals Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin.

1991 ◽  
Vol 115 (1) ◽  
pp. 267-277 ◽  
Author(s):  
S P Kennedy ◽  
S L Warren ◽  
B G Forget ◽  
J S Morrow
Keyword(s):  
Binding Site ◽  
Binding Capacity ◽  
Repeat Unit ◽  
Expression System ◽  
Membrane Vesicles ◽  
Enzymatic Digestion ◽  
Binding Domain ◽  
Amino Terminal ◽  
Prokaryotic Expression System

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.

Production of recombinant IGF1 and its action on neuroblastoma cells in vitro

2018 ◽  
Vol 18 (4) ◽  
pp. 34-41
Author(s):  
Sergey A. Ishuk ◽  
Elena G. Bogomolova ◽  
Olga A. Dobrovolskaya ◽  
Alyona O. Akhmetshina ◽  
Daria S. Krasnoshchek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Cation Exchange ◽  
Expression System ◽  
Neuroblastoma Cells ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Recombinant Insulin ◽  
Prokaryotic Expression System

This study aimed to develop a method for producing human recombinant insulin-like growth factor (IGF-1) based on a prokaryotic expression system and to characterize the highly purified protein. To achieve the study’s goal, the following methods were conducted: we performed automated chemical synthesis of DNA, constructed the expression plasmid, obtained Escherichia coli cell-producers of human recombinant IGF-1, cultivated the obtained producer cells with the induction of recombinant protein synthesis by isopropyl-β-D-1-thiogalactopyranoside and lactose, and purified human recombinant IGF-1 with affinity and cation exchange chromatography. The recombinant protein IGF-1 forms inclusion bodies during synthesis in Escherichia coli BL21 cells that contain plasmid pET28-IGF-1. Purified recombinant protein was obtained with a purity of 98% using affinity and cation exchange chromatography methods. The protein yield was 6 mg of human recombinant IGF-1 from 1 g of raw biomass. The resulting protein has the ability to protect Neuro 2a neuroblastoma cells from death caused by the deprivation of serum in the culture medium and can stimulate the differentiation of cells into neurons. Thus, a highly purified human recombinant IGF-1 was obtained. This protein has biological activity and is suitable for preclinical studies.

scholarly journals Pancreatic Amylase Is an Environmental Signal for Regulation of Biofilm Formation and Host Interaction in Campylobacter jejuni

2015 ◽  
Vol 83 (12) ◽  
pp. 4884-4895 ◽  
Author(s):  
Waheed Jowiya ◽  
Katja Brunner ◽  
Sherif Abouelhadid ◽  
Haitham A. Hussain ◽  
Sean P. Nair ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Pancreatic Amylase ◽  
Content Type ◽  
Host Interaction ◽  
Food Borne ◽  
Epithelial Cell Lines

Campylobacter jejuniis a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran byC. jejuniand that a secreted protease, Cj0511, is required. Exposure ofC. jejunito pancreatic amylase promotes biofilm formationin vitro, increases interaction with human epithelial cell lines, increases virulence in theGalleria mellonellainfection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protectsC. jejunifrom stress conditionsin vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.

scholarly journals Preliminary study on the application of PspA as carrier

2018 ◽  
Author(s):  
Lichan-Wang ◽  
Yajun-Tan ◽  
Chen-Wei ◽  
Huajie-Zhang ◽  
Peng-Luo ◽  
...  
Keyword(s):  
Expression System ◽  
Protein A ◽  
Surface Protein ◽  
Positive Control ◽  
Surface Proteins ◽  
Carrier Protein ◽  
Streptococcus Pneumonia ◽  
Protein Conjugates ◽  
Prokaryotic Expression System ◽  
Group A

AbstractThe aim of the study is to research the feasibility of pneumococcal surface protein A (PspA) using as carrier protein. Three recombinant pneumococcal surface proteins A (come from family1 and family 2) were expressed by prokaryotic expression system and were conjugated to group A meningococcal polysaccharide (GAMP) to make three polysaccharide-protein conjugates. The conjugates, un-conjugated proteins, GAMP and GAMP-TT vaccine bulk (used as positive control) were immunized to mice and their immune effects were evaluated by the method of ELISA, FCM and SBA. The results showed that the polysaccharide-protein conjugates can produce higher levels of anti-GAMP IgG titers (P < 0.05), higher ratios of Th1/Th2 (P < 0.05) and higher levels of serum bactericidal activity (P < 0.05) compared with the un-conjugated GAMP. The conjugation of PspAs to GAMP also enhanced the anti-PspA responses compared with un-conjugated PspAs except PspA3. In conclusion, all the results indicated that three PspAs were suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the GAMP, and would protect against group A of epidemic cerebrospinal meningitis and also have the potential to provide broad protection from Streptococcus pneumonia.

In Silico Design of a Novel Multi-Epitope Peptide Vaccine Against Hepatocellular Carcinoma

2020 ◽  
Vol 17 (9) ◽  
pp. 1164-1176
Author(s):  
Fatemeh Motamedi Dehbarez ◽  
Navid Nezafat ◽  
Shirin Mahmoodi
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Lymphocytes ◽  
Immune Responses ◽  
Tandem Repeats ◽  
3D Structure ◽  
Peptide Vaccine ◽  
Humoral Immune ◽  
Innovative Therapy

Background: Hepatocellular Carcinoma (HCC) is a prevalent cancer in the world. As yet, there is no medication for complete treatment of HCC. Objective: There is a critical need to search for an innovative therapy for HCC. Recently, multiepitope vaccines have been introduced as effective immunotherapy approach against HCC. Methods: In this research, several immunoinformatics methods were applied to create an original multi-epitope vaccine against HCC consisting of CD8+ cytolytic T lymphocytes (CTLs) epitopes selected from α- fetoprotein (AFP), glypican-3 (GPC3), aspartyl-β-hydroxylase (ASPH); CD4+ helper T lymphocytes (HTLs) epitopes from tetanus toxin fragment C (TTFC), and finally, two tandem repeats of HSP70407-426 were used which stimulated strong innate and adaptive immune responses. All the mentioned parts were connected together by relevant linkers. Results: According to physicochemical, structural, and immunological results, the designed vaccine is stable, non-allergen, antigen; it also has a high-quality 3D structure, and numerous linear and conformational B cell epitopes, whereby this vaccine may stimulate efficient humoral immunity. Conclusion: Center on the collected results, the designed vaccine potentially can induce cellular and humoral immune responses in HCC cases; nonetheless, the efficiency of vaccine must be approved within in vitro and in vivo immunological analyzes.

scholarly journals Five-Member Gene Family of Bartonella quintana

2003 ◽  
Vol 71 (2) ◽  
pp. 814-821 ◽  
Author(s):  
Michael F. Minnick ◽  
Kate N. Sappington ◽  
Laura S. Smitherman ◽  
Siv G. E. Andersson ◽  
Olof Karlberg ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Bartonella Henselae ◽  
Family Members ◽  
Surface Protein ◽  
Etiologic Agent ◽  
The Other ◽  
Rt Pcr ◽  
Transcription Analysis ◽  
Bartonella Quintana

ABSTRACT Bartonella quintana, the agent of trench fever and an etiologic agent of bacillary angiomatosis, has an extraordinarily high hemin requirement for growth compared to other bacterial pathogens. We previously identified the major hemin receptor of the pathogen as a 30-kDa surface protein, termed HbpA. This report describes four additional homologues that share approximately 48% amino acid sequence identity with hbpA. Three of the genes form a paralagous cluster, termed hbpCAB, whereas the other members, hbpD and hbpE, are unlinked. Secondary structure predictions and other evidence suggest that Hbp family members are β-barrels located in the outer membrane and contain eight transmembrane domains plus four extracellular loops. Homologs from a variety of gram-negative pathogens were identified, including Bartonella henselae Pap31, Brucella Omp31, Agrobacterium tumefaciens Omp25, and neisserial opacity proteins (Opa). Family members expressed in vitro-synthesized proteins ranging from ca. 26.5 to 35.1 kDa, with the exception of HbpB, an ∼55.9-kDa protein whose respective gene has been disrupted by a ∼510 GC-rich element containing variable-number tandem repeats. Transcription analysis by quantitative reverse transcriptase-PCR (RT-PCR) indicates that all family members are expressed under normal culture conditions, with hbpD and hbpB transcripts being the most abundant and the rarest, respectively. Mutagenesis of hbpA by allelic exchange produced a strain that exhibited an enhanced hemin-binding phenotype relative to the parental strain, and analysis by quantitative RT-PCR showed elevated transcript levels for the other hbp family members, suggesting that compensatory expression occurs.

scholarly journals Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray

2020 ◽  
Author(s):  
Tate Oulton ◽  
Joshua Obiero ◽  
Isabel Rodriguez ◽  
Isaac Ssewanyana ◽  
Rebecca A Dabbs ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Microarray ◽  
Expression System ◽  
Protein Microarrays ◽  
In Vitro Transcription ◽  
Antibody Responses ◽  
Dependent Manner ◽  
Expression Systems ◽  
Protein Antigens

The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro Transcription/Translation (IVTT) systems (a similarly high-throughput protein expression method) are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on a protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from opposing expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clearly defined relationship between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches.

scholarly journals The therapeutic effect of a novel anti-TNF-α/IL-6R triple-specific fusion protein under experimental septic condition

2021 ◽  
Author(s):  
Xiaole Chen ◽  
Shuangyu Tan ◽  
Mengru Yan ◽  
Kaimei Nie ◽  
Qingmei Zheng ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Expression System ◽  
Cecal Ligation ◽  
Mouse Fibroblast ◽  
Potential Candidate ◽  
Tnf Α ◽  
Prokaryotic Expression System

Abstract A novel anti-TNF-α/IL-6R triple-specific fusion protein, by linking 3 single domain chains, was designed and constructed in our lab. The high purity fusion proteins were obtained by our developed prokaryotic expression system process with high binding affinity with TNF-α (94.75 pM), Human Serum Albumin (1.83 nM) and IL-6R (2.29 nM). In this study, the anti-TNF-α/IL-6R triple-specific fusion protein protected the mouse fibroblast fibrosarcoma cell line (L929) from the apoptosis effects induced by TNF-α, establishing that the expressed fusion proteins can selectively combine with TNF-α in vitro. In vivo, the survival rate of cecal ligation and puncture (CLP) was notably increased in the group with anti-TNF-α/IL-6R triple-specific fusion protein treatment, and meaningfully higher compared with the single-targeted IL-6R and TNF-α fusion protein at the same dose. After the treatment with anti-TNF-α/IL-6R triple-specific fusion protein, the level of serum TNF-α, IL-1β and IL-6 were significantly decreased, and sepsis-induced pathological injuries in the kidney were remarkably attenuated. The anti-TNF-α/IL-6R triple-specific fusion protein can be the potential candidate for the development of new drug design against sepsis.

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