scholarly journals Designing of DNA Vaccine Based on a Secretory Form of Major Capsid Protein of Human Papillomavirus Type 18

2021 ◽  
Author(s):  
Somayeh Shokri ◽  
Shahab Mahmoudvand ◽  
Manoochehr Makvandi ◽  
Reza Taherkhani ◽  
Mohammad Rashno ◽  
...  
Keyword(s):  
Western Blot ◽  
Vaccine Development ◽  
Restriction Enzymes ◽  
Eukaryotic Expression ◽  
Human Papillomaviruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hpv Vaccines ◽  
Hek 293 ◽  
Colony Pcr ◽  
Low Middle Income Countries

  More than 99% of cervical cancers are associated with human papillomaviruses (HPVs) worldwide. Current HPV vaccines are safe, highly immunogenic, with effective immunity against specific HPV types. However, DNA vaccines are a new appealing platform which can be considered for designing the HPV vaccines. This study aimed to construct a recombinant eukaryotic expression plasmid containing L1 of HPV-18, tissue plasminogen activators (tPA), and pan HLA DR-binding epitope (PADRE) genes into the pVAX1 vector. The L1, tPA, and PADRE genes were amplified in a thermocycler. The polymerase chain reaction (PCR) products were cloned and insertion of the genes was confirmed using colony PCR, restriction enzymes analysis, and sequencing methods. Indirect immunofluorescence, RT-PCR, and western blot assays were applied to identify the target gene in HEK-293 cells. Total IgG and its isotypes in immunized mice were measured by enzyme-linked immunosorbent assay technique. Western blot analysis showed a protein band of about 67.5 kDa in supernatant and cell lysate of transfected cells. The results of mice immunization with different constructs (group 1: the pVAX-L1, group 2: pVAX-tPA-PADRE-L1, group 3: pVAX1, and group 4: PBS as controls) indicated that the pVAX1-tPA-PADRE-L1 construct induced a significantly higher level of total IgG than pVAX1-L1 (p=0.003). In conclusion, pVAX1-tPA-PADRE-L1 recombinant plasmid is a highly immunogenic construct and suggests as a promising candidate for vaccine development against HPV type 18 in low-middle-income countries.  

scholarly journals Advances in the study of nodavirus

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3841 ◽  
Author(s):  
Chean Yeah Yong ◽  
Swee Keong Yeap ◽  
Abdul Rahman Omar ◽  
Wen Siang Tan
Keyword(s):  
Reverse Transcription ◽  
Vaccine Development ◽  
Eukaryotic Expression ◽  
Oral Feeding ◽  
Viral Rna ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nucleotide Sequencing ◽  
Type I ◽  
Mechanism Study ◽  
The Caspian Sea

Nodaviruses are small bipartite RNA viruses which belong to the family ofNodaviridae. They are categorized into alpha-nodavirus, which infects insects, and beta-nodavirus, which infects fishes. Another distinct group of nodavirus infects shrimps and prawns, which has been proposed to be categorized as gamma-nodavirus. Our current review focuses mainly on recent studies performed on nodaviruses. Nodavirus can be transmitted vertically and horizontally. Recent outbreaks have been reported in China, Indonesia, Singapore and India, affecting the aquaculture industry. It also decreased mullet stock in the Caspian Sea. Histopathology and transmission electron microscopy (TEM) are used to examine the presence of nodaviruses in infected fishes and prawns. For classification, virus isolation followed by nucleotide sequencing are required. In contrast to partial sequence identification, profiling the whole transcriptome using next generation sequencing (NGS) offers a more comprehensive comparison and characterization of the virus. For rapid diagnosis of nodavirus, assays targeting the viral RNA based on reverse-transcription PCR (RT-PCR) such as microfluidic chips, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and RT-LAMP coupled with lateral flow dipstick (RT-LAMP-LFD) have been developed. Besides viral RNA detections, diagnosis based on immunological assays such as enzyme-linked immunosorbent assay (ELISA), immunodot and Western blotting have also been reported. In addition, immune responses of fish and prawn are also discussed. Overall, in fish, innate immunity, cellular type I interferon immunity and humoral immunity cooperatively prevent nodavirus infections, whereas prawns and shrimps adopt different immune mechanisms against nodavirus infections, through upregulation of superoxide anion, prophenoloxidase, superoxide dismutase (SOD), crustin, peroxinectin, anti-lipopolysaccharides and heat shock proteins (HSP). Potential vaccines for fishes and prawns based on inactivated viruses, recombinant proteins or DNA, either delivered through injection, oral feeding or immersion, are also discussed in detail. Lastly, a comprehensive review on nodavirus virus-like particles (VLPs) is presented. In recent years, studies on prawn nodavirus are mainly focused onMacrobrachium rosenbergiinodavirus (MrNV). RecombinantMrNV VLPs have been produced in prokaryotic and eukaryotic expression systems. Their roles as a nucleic acid delivery vehicle, a platform for vaccine development, a molecular tool for mechanism study and in solving the structures ofMrNV are intensively discussed.

scholarly journals Immunogenicity of a plasmid DNA vaccine encoding G1 epitope of bovine ephemeral fever virus G glycoprotein in mice

2018 ◽  
Vol 85 (1) ◽  
Author(s):  
Reza Pasandideh ◽  
Masoud Reza Seyfi Abad Shapouri ◽  
Mohammad Taghi Beigi Nassiri
Keyword(s):  
Dna Vaccine ◽  
Plasmid Dna ◽  
Eukaryotic Expression ◽  
Fever Virus ◽  
Immunofluorescent Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Ephemeral Fever Virus ◽  
Hek 293 ◽  
Specific Antibodies ◽  
Bovine Ephemeral Fever

The aim of this study was to investigate the immunogenicity of a plasmid deoxyribonucleic acid (DNA) vaccine encoding the G1 epitope of bovine ephemeral fever virus (BEFV) G glycoprotein in mice. A plasmid DNA carrying the G1 gene was constructed and designated as pcDNA3.1-G1. The expression of the target gene was confirmed in human embryonic kidney 293 (HEK 293) cells transfected with pcDNA3.1-G1 by indirect immunofluorescent staining. Immunisation experiments were intramuscularly carried out by vaccinating 6-week-old female mice in four groups, including the pcDNA3.1-G1 construct, pcDNA3.1 (+) plasmid alone, BEF-inactivated vaccine and phosphate-buffered saline (PBS) (1X) three times with 2-week intervals. Fourteen days after the last immunisation, the animals were bled and the resulting sera were tested for anti-G1-specific antibodies by immunoblotting analysis, indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralisation (VN) test. Serological assays showed that the pcDNA3.1-G1 construct expressing G1 protein was able to elicit specific antibodies against this antigen. Virus neutralisation test showed that pcDNA3.1-G1 could induce anti-BEFV-neutralising antibodies in mice. Our findings indicated that a new dimension can be added to vaccine studies for bovine ephemeral fever (BEF) using eukaryotic expression plasmids encoding the G1 antigen in the future.

scholarly journals The Alterations in Methylene Blue/Light-Treated Frozen Plasma Proteins Revealed by Proteomics

2021 ◽  
pp. 1-8
Author(s):  
Tiange Wu ◽  
Xiaoning Wang ◽  
Kai Ren ◽  
Xiaochen Huang ◽  
Jiankai Liu
Keyword(s):  
Mass Spectrometry ◽  
Methylene Blue ◽  
Western Blot ◽  
Blue Light ◽  
Differentially Expressed ◽  
Enzyme Linked Immunosorbent Assay ◽  
Differentially Expressed Protein ◽  
Significant Difference ◽  
Modified Proteins ◽  
Frozen Plasma

Introduction: The aim of this study was to investigate the modified proteins in methylene blue/light-treated frozen plasma (MB-FP) compared with fresh frozen plasma (FFP) in order to gain a better application of MB/light-treated plasma in clinic transfusion. Methods: MB-FP and FFP were collected from Changchun central blood station, and a trichloroacetic acid/acetone precipitation method was used to remove albumin for the enrichment of lower abundance proteins. The plasma protein in MB-FP and FFP were separated using two-dimensional gel electrophoresis (2-DE) and the differentially expressed protein spots were analyzed using mass spectrometry. Finally, the differentially expressed proteins were tested using Western blot and enzyme-linked immunosorbent assay (ELISA). Results: Approximately 14 differentially expressed protein spots were detected in the MB-FP, and FFP was chosen as the control. After 2-DE comparison analysis and mass spectrometry, 8 significantly differentially expressed protein spots were identified, corresponding to 6 different proteins, including complement C1r subcomponent (C1R), inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4), keratin, type II cytoskeletal 1 (KRT1), hemopexin (HPX), fibrinogen gamma chain (FGG), and transthyretin (TTR). Western blot showed no significant difference in the expression level of KRT1 between MB-FP and FFP (p > 0.05). Both Western blot and ELISA indicated that the level of HPX was significantly higher in FFP than in MB-FP (p < 0.05). Conclusion: This comparative proteomics study revealed that some significantly modified proteins occur in MB-FP, such as C1R, ITI-H4, KRT1, HPX, FGG, and TTR. Our findings provide more theoretical data for using MB-FP in transfusion medicine. However, the relevance of the data for the transfusion of methylene blue/light-treated plasma remains unclear. The exact modification of these proteins and the effects of these modified proteins on their functions and their effects in clinical plasma infusion need to be further studied.

scholarly journals Mesenchymal stem cell-originated exosomal lncRNA HAND2-AS1 impairs rheumatoid arthritis fibroblast-like synoviocyte activation through miR-143-3p/TNFAIP3/NF-κB pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Yuhua Su ◽  
Yajing Liu ◽  
Chao Ma ◽  
Chunxiao Guan ◽  
Xiufen Ma ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Tumor Necrosis Factor ◽  
Western Blot ◽  
Tumor Necrosis ◽  
Cell Counting ◽  
Biological Behavior ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Interaction ◽  
Necrosis Factor

Abstract Background Long non-coding RNA heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) was found to be elevated in rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLSs). However, whether HAND2-AS1 functions as an exosomal lncRNA related to mesenchymal stem cells (MSCs) in RA progression is unknown. Methods The expression of HAND2-AS1, microRNA (miR)-143-3p, and tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) was detected using quantitative real-time polymerase chain reaction and Western blot. Cell proliferation, apoptosis, migration, and invasion were detected using cell counting kit-8, flow cytometry, and wound healing and transwell assays. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL)-6 were analyzed using enzyme-linked immunosorbent assay. The level of phosphorylated-p65 was examined by Western blot. The binding interaction between miR-143-3p and HAND2-AS1 or TNFAIP3 was confirmed by the dual-luciferase reporter and RIP assays. Exosomes were isolated by ultracentrifugation and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Results HAND2-AS1 was lowly expressed in RA synovial tissues, and HAND2-AS1 re-expression suppressed the proliferation, motility, and inflammation and triggered the apoptosis in RA-FLSs via the inactivation of NF-κB pathway. Mechanistically, HAND2-AS1 directly sponged miR-143-3p and positively regulated TNFAIP3 expression, the target of miR-143-3p. Moreover, the effects of HAND2-AS1 on RA-FLSs were partially attenuated by miR-143-3p upregulation or TNFAIP3 knockdown. HAND2-AS1 could be packaged into hMSC-derived exosomes and absorbed by RA-FLSs, and human MSC-derived exosomal HAND2-AS1 also repressed above malignant biological behavior of RA-FLSs. Conclusion MSC-derived exosomes participated in the intercellular transfer of HAND2-AS1 and suppressed the activation of RA-FLSs via miR-143-3p/TNFAIP3/NF-κB pathway, which provided a novel insight into the pathogenesis and treatment of RA.

scholarly journals NEAT1 aggravates sepsis-induced acute kidney injury by sponging miR-22-3p

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 333-342
Author(s):  
Yawei Feng ◽  
Jun Liu ◽  
Ranliang Wu ◽  
Peng Yang ◽  
Zhiqiang Ye ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Cell Injury ◽  
Kidney Injury ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay

AbstractBackground and aimAcute kidney injury (AKI) is a common complication of sepsis. Long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) plays a vital role in various diseases, including AKI. This study aimed to investigate the function and mechanism of NEAT1 in sepsis-induced AKI.Materials and methodsA septic AKI model was established by treating HK-2 cells with lipopolysaccharide (LPS). The levels of NEAT1 and miR-22-3p were measured by quantitative real-time PCR. Cell apoptosis was assessed by flow cytometry. The levels of apoptosis-related protein and autophagy-related factors were examined by the western blot assay. An enzyme-linked immunosorbent assay was used to calculate the contents of inflammatory factors. The interaction between NEAT1 and miR-22-3p was validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA pull-down assay. The levels of nuclear factor (NF)-κB pathway-related proteins were evaluated by the western blot assay.ResultsNEAT1 was upregulated, while miR-22-3p was downregulated in patients with sepsis and in LPS-stimulated HK-2 cells. LPS treatment triggered cell apoptosis, autophagy, and inflammatory response in HK-2 cells. NEAT1 knockdown attenuated LPS-induced cell injury. NEAT1 modulated LPS-triggered cell injury by targeting miR-22-3p. Furthermore, NEAT1 regulated the NF-κB pathway by modulating miR-22-3p.ConclusionDepletion of NEAT1 alleviated sepsis-induced AKI via regulating the miR-22-3p/NF-κB pathway.

scholarly journals Duck RIG-I CARD Domain Induces the Chicken IFN-βby Activating NF-κB

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Yang Chen ◽  
Zhengyang Huang ◽  
Bin Wang ◽  
Qinming Yu ◽  
Ran Liu ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Eukaryotic Expression ◽  
Poly I:C ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Regulatory Domains ◽  
Polycytidylic Acid ◽  
Card Domain ◽  
Inducible Gene

Retinoic acid-inducible gene I- (RIG-I-) like receptors (RLRs) have recently been identified as cytoplasmic sensors for viral RNA. RIG-I, a member of RLRs family, plays an important role in innate immunity. Although previous investigations have proved that RIG-I is absent in chickens, it remains largely unknown whether the chicken can respond to RIG-I ligand. In this study, the eukaryotic expression vectors encoding duRIG-I full length (duck RIG-I, containing all domains), duRIG-I N-terminal (containing the two caspase activation and recruitment domain, CARDs), and duRIG-I C-terminal (containing helicase and regulatory domains) labeled with 6*His tags were constructed successfully and detected by western blotting. Luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA) detected the duRIG-I significantly activated NF-κB and induced the expression of IFN-βwhen polyinosinic-polycytidylic acid (poly[I:C], synthetic double-stranded RNA) challenges chicken embryonic fibroblasts cells (DF1 cells), while the duRIG-I was inactive in the absence of poly[I:C]. Further analysis revealed that the CARDs (duRIG-I-N) induced IFN-βproduction regardless of the presence of poly[I:C], while the CARD-lacking duRIG-I (duRIG-I-C) was not capable of activating downstream signals. These results indicate that duRIG-I CARD domain plays an important role in the induction of IFN-βand provide a basis for further studying the function of RIG-I in avian innate immunity.

In silico and in vitro analysis of a multiepitope L1-E7 fusion construct for vaccine development against human papillomaviruses

2021 ◽  
Vol 18 ◽  
Author(s):  
Elnaz Abbasifarid ◽  
Azam Bolhassani ◽  
Shiva Irani ◽  
Fattah Sotoodehnejadnematalahi
Keyword(s):  
Western Blotting ◽  
Vaccine Development ◽  
Fluorescent Microscopy ◽  
Therapeutic Vaccine ◽  
Human Papillomaviruses ◽  
Mhc I ◽  
Fusion Construct ◽  
Mhc Ii ◽  
Hpv Types

Background: Human papillomavirus (HPV) infection is the major risk factor for cervical cancer. Current prophylactic HPV vaccines provide immunity against most genital and carcinogenic HPV types. However, these vaccines failed to produce immune responses against already established HPV infections. Methods: For the design of a therapeutic vaccine candidate, we utilized immunoinformatics tools to design a potential multiepitope fusion construct based on L1 and E7 genes from different high- and low-risk HPV types. After determination of CD4+ and CD8+ T cell epitopes, the allergenicity, toxicity, immunogenicity, conservancy, and population coverage were analyzed for epitope selection. Then, the hemolytic probability of the selected epitopes, and molecular docking between major histocompatibility complex (MHC) and the chosen epitopes were performed by different web servers. Next, a multiepitope peptide construct consisting of 12 epitopes linked by the AAY proteasomal sequence was designed. After that, physicochemical properties, solubility, secondary and tertiary structures of this construct were evaluated by bioinformatics tools. Finally, after amino acid reverse translation of the multiepitope peptide construct, expression of the L1-E7 DNA construct (pEGFP-L1-E7) was investigated in HEK-293T cells using fluorescent microscopy, flow cytometry, and western blotting. Results: Considering various parameters, the immunodominant peptides such as L1(MHC-I)-DLDQFPLGRKFLLQ, L1(MHC-II)-NQLFVTVVDTTRSTN, E7-HPV16(MHC-I)-AEPDRAHYNIVTF, E7-HPV18(MHC-I)-HGPKATVQDIVLHL, E7-HPV31(MHC-I)-KPDTSNYNIVTF, E7-HPV33(MHC-I)-RPDGQAQPATADYYI, E7-HPV45(MHC-I)- RTLQQLFLSFV, E7-HPV16(MHC-II)-TLHEYMLDLQPETTD, E7-HPV18(MHC-II)-LRAFQQLFLNTLSFV, E7-HPV31(MHC-II)-PTLQDYVLDLQPEAT, E7-HPV33(MHC-II)-LKEYVLDLYPEPTDL and E7-HPV45(MHC-II)-LQQLFLSTLSFVCPW were determined to design the vaccine construct. The results indicated efficient expression of the L1-E7 DNA construct (74 ± 2.19%) in vitro. Moreover, the polyepitope peptide generated in the cells was detected as a clear band of ~ 50 kDa in western blotting. Conclusion: Regarding the favorable transfection efficiency of the designed L1-E7 multiepitope construct, in vivo validation study on its therapeutic potential is underway.

scholarly journals Pharmacological Basis for Use of a Novel Compound in Hyperuricemia: Anti-Hyperuricemic and Anti-Inflammatory Effects

2021 ◽  
Vol 12 ◽  
Author(s):  
Lei Zhao ◽  
Yihang Li ◽  
Dahong Yao ◽  
Ran Sun ◽  
Shifang Liu ◽  
...  
Keyword(s):  
Uric Acid ◽  
Western Blot ◽  
Serum Uric Acid ◽  
Glucose Transporter ◽  
Serum Uric Acid Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Mice Model ◽  
Protein Levels ◽  
Anti Inflammatory

Background: The prevalence of hyperuricemia is considered high worldwide. Hyperuricemia occurs due to decreased excretion of uric acid, increased synthesis of uric acid, or a combination of both mechanisms. There is growing evidence that hyperuricemia is associated with a decline of renal function.Purpose: This study is aimed at investigating the effects of the novel compound on lowering the serum uric acid level and alleviating renal inflammation induced by high uric acid in hyperuricemic mice.Methods: Hyperuricemic mice model was induced by potassium oxonate and used to evaluate the effects of the novel compound named FxUD. Enzyme-linked immunosorbent assay was used to detect the related biochemical markers. Hematoxylin-eosin (HE) staining was applied to observe pathological changes. The mRNA expression levels were tested by qRT-PCR. The protein levels were determined by Western blot. In parallel, human proximal renal tubular epithelial cells (HK-2) derived from normal kidney was used to further validate the anti-inflammatory effects in vitro.Results: FxUD administration significantly decreased serum uric acid levels, restored the kidney function parameters, and improved the renal pathological injury. Meanwhile, treatment with FxUD effectively inhibited serum and liver xanthine oxidase (XOD) levels. Reversed expression alterations of renal inflammatory cytokines, urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) were observed in hyperuricemic mice. Western blot results illustrated FxUD down-regulated protein levels of inflammasome components. Further studies showed that FxUD inhibited the activation of NF-κB signaling pathway in the kidney of hyperuricemic mice. In parallel, the anti-inflammatory effect of FxUD was also confirmed in HK-2.Conclusion: Our study reveals that FxUD exhibits the anti-hyperuricemic and anti-inflammatory effects through regulating hepatic XOD and renal urate reabsorption transporters, and suppressing NF-κB/NLRP3 pathway in hyperuricemia. The results provide the evidence that FxUD may be potential for the treatment of hyperuricemia with kidney inflammation.

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