scholarly journals Erythrocyte Adhesion of Merozoite Surface Antigen 2c1 Expressed During Extracellular Stages of Babesia orientalis

2021 ◽  
Vol 12 ◽  
Author(s):  
Zheng Nie ◽  
Yangsiqi Ao ◽  
Sen Wang ◽  
Xiang Shu ◽  
Muxiao Li ◽  
...  
Keyword(s):  
Surface Antigen ◽  
Water Buffalo ◽  
Expression System ◽  
Infectious Agent ◽  
Polyclonal Antiserum ◽  
Eukaryotic Expression ◽  
Effective Vaccine ◽  
Babesia Orientalis ◽  
Merozoite Surface Antigen

Babesia orientalis, a major infectious agent of water buffalo hemolytic babesiosis, is transmitted by Rhipicephalus haemaphysaloides. However, no effective vaccine is available. Essential antigens that are involved in parasite invasion of host red blood cells (RBCs) are potential vaccine candidates. Therefore, the identification and the conduction of functional studies of essential antigens are highly desirable. Here, we evaluated the function of B. orientalis merozoite surface antigen 2c1 (BoMSA-2c1), which belongs to the variable merozoite surface antigen (VMSA) family in B. orientalis. We developed a polyclonal antiserum against the purified recombinant (r)BoMSA-2c1 protein. Immunofluorescence staining results showed that BoMSA-2c1 was expressed only on extracellular merozoites, whereas the antigen was undetectable in intracellular parasites. RBC binding assays suggested that BoMSA-2c1 specifically bound to buffalo erythrocytes. Cytoadherence assays using a eukaryotic expression system in vitro further verified the binding and inhibitory ability of BoMSA-2c1. We found that BoMSA-2c1 with a GPI domain was expressed on the surface of HEK293T cells that bound to water buffalo RBCs, and that the anti-rBoMSA2c1 antibody inhibited this binding. These results indicated that BoMSA-2c1 was involved in mediating initial binding to host erythrocytes of B. orientalis. Identification of the occurrence of binding early in the invasion process may facilitate understanding of the growth characteristics, and may help in formulating strategies for the prevention and control of this parasite.

scholarly journals Cellular encoding of Cy dyes for single-molecule imaging

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lilia Leisle ◽  
Rahul Chadda ◽  
John D Lueck ◽  
Daniel T Infield ◽  
Jason D Galpin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Cyanine Dye ◽  
Nonsense Suppression ◽  
Xenopus Laevis Oocyte ◽  
Cellular Environment ◽  
Oocyte Expression System ◽  
Improved Technique

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.

Antibodies induced by Plasmodium falciparum merozoite surface antigen-2-designed pseudopeptides possess neutralizing properties of the in vitro malarial infection

Peptides ◽  
2007 ◽  
Vol 28 (10) ◽  
pp. 1954-1965 ◽  
Author(s):  
José Manuel Lozano ◽  
Francy J. Montoya-Fajardo ◽  
Johan Hoebeke ◽  
Gladys H. Cifuentes ◽  
Martha Forero ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Antigen ◽  
Malarial Infection ◽  
Merozoite Surface Antigen ◽  
Falciparum Merozoite

scholarly journals Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

2021 ◽  
Vol 17 (7) ◽  
pp. e1008864
Author(s):  
Duncan N. Ndegwa ◽  
Prasun Kundu ◽  
Jessica B. Hostetler ◽  
Alejandro Marin-Menendez ◽  
Theo Sanderson ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Culture System ◽  
Vaccine Development ◽  
Expression System ◽  
Critical Role ◽  
Blood Stage ◽  
Effective Vaccine ◽  
Mammalian Expression ◽  
Close Phylogenetic Relationship

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.

scholarly journals Glycosylation Tunes Neuroserpin Physiological and Pathological Properties

2020 ◽  
Vol 21 (9) ◽  
pp. 3235 ◽  
Author(s):  
Cristina Visentin ◽  
Luca Broggini ◽  
Benedetta Maria Sala ◽  
Rosaria Russo ◽  
Alberto Barbiroli ◽  
...  
Keyword(s):  
Pathological Condition ◽  
Expression System ◽  
Point Mutations ◽  
Tryptophan Fluorescence ◽  
Biochemical Properties ◽  
Eukaryotic Expression ◽  
Cell Models ◽  
Wild Type

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

Delivery of HIV-1 Polyepitope Constructs Using Cationic and Amphipathic Cell Penetrating Peptides into Mammalian Cells

2020 ◽  
Vol 17 (6) ◽  
pp. 408-428 ◽  
Author(s):  
Fatemeh Namazi ◽  
Azam Bolhassani ◽  
Seyed Mehdi Sadat ◽  
Shiva Irani
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
293T Cell ◽  
Cell Penetrating Peptides ◽  
Expression Vectors ◽  
Effective Vaccine ◽  
In Silico Studies ◽  
Cell Penetrating ◽  
Hiv 1

Background: An effective vaccine against human immunodeficiency virus 1 (HIV-1) is an important global health priority. Despite many efforts in the development of the HIV-1 vaccine, no effective vaccine has been approved yet. Recently, polyepitope vaccines including several immunogenic and conserved epitopes of HIV-1 proteins have received special attention. Methods: In this study, HIV-1 Nef, Tat, Gp160 and P24 proteins were considered for selection of immunodominant and conserved epitopes due to their critical roles in the viral life cycle and pathogenesis. At first, the Nef60-84-Nef126-144-Tat29-49-Gp16030-53-Gp160308-323-P248-151 DNA construct was designed using in silico studies. Then, the DNA construct was subcloned in pEGFP-N1 and pET- 24a (+) expression vectors and the rNef-Tat-Gp160-P24 polyepitope peptide was generated in E.coli expression system for in vitro delivery using novel cell-penetrating peptides (CPPs), LDP-NLS and CyLoP-1, in a non-covalent manner. Also, the HR9 and MPG CPPs were used to transfer the DNA construct. Results: Our results showed that the recombinant polyepitope peptide generated in Rosetta strain migrated as a clear band of ~31 kDa in SDS-PAGE. The SEM data confirmed the formation of stable nanoparticles with a size below 250 nm. MTT assay revealed that the complexes did not represent any considerable cytotoxic effect compared to untreated cells. The results of fluorescence microscopy, flow cytometry and western blotting indicated that these CPPs successfully delivered polyepitope constructs into HEK-293T cell line. Conclusion: These data suggested that these CPPs can be used as a promising approach for the development of the HIV-1 vaccine.

scholarly journals Construction of a eukaryotic expression system for granulysin and its protective effect in mice infected with Mycobacterium tuberculosis

2006 ◽  
Vol 55 (10) ◽  
pp. 1389-1393 ◽  
Author(s):  
Binbo Liu ◽  
Shengwu Liu ◽  
Xueju Qu ◽  
Junyan Liu
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protective Effect ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Control Group ◽  
Eukaryotic Expression Plasmid ◽  
Expression Plasmid ◽  
Eukaryotic Expression System ◽  
Viable Bacteria

A full-length cDNA of granulysin was inserted into the pcDNA3.1(−) vector to construct a eukaryotic expression plasmid for granulysin. The recombinant plasmids were injected intramuscularly into mice infected with Mycobacterium tuberculosis to evaluate the protective effect of granulysin. Granulysin significantly decreased the weight index (WI) of the spleen, reduced the numbers of viable bacteria in lung and spleen, and reduced the lesions of lung tissue in granulysin-rDNA-immunized mice compared with those of control group mice. In vitro, the serum of the recombinant-plasmid-immunized mice inhibited the viability of M. tuberculosis by the physical disruption of cell membranes. Therefore, granulysin has a therapeutic effect against M. tuberculosis.

Characterization of the variable merozoite surface antigen (VMSA) gene family of Babesia orientalis

2020 ◽  
Vol 119 (11) ◽  
pp. 3639-3648
Author(s):  
Zheng Nie ◽  
Yingjun Xia ◽  
Long Yu ◽  
Muxiao Li ◽  
Jiaying Guo ◽  
...  
Keyword(s):  
Gene Family ◽  
Surface Antigen ◽  
Babesia Orientalis ◽  
Merozoite Surface Antigen

scholarly journals Babesia bovis Merozoite Surface Antigen 1 and Rhoptry-Associated Protein 1 Are Expressed in Sporozoites, and Specific Antibodies Inhibit Sporozoite Attachment to Erythrocytes

2002 ◽  
Vol 70 (3) ◽  
pp. 1599-1603 ◽  
Author(s):  
Juan Mosqueda ◽  
Terry F. McElwain ◽  
David Stiller ◽  
Guy H. Palmer
Keyword(s):  
Surface Antigen ◽  
Babesia Bovis ◽  
Specific Antibodies ◽  
Erythrocyte Invasion ◽  
Merozoite Surface Antigen

ABSTRACT We examined Babesia bovis sporozoites for the expression of two molecules, merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), that are postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites. Importantly, monospecific MSA-1 and RAP-1 antisera each inhibited sporozoite invasion of erythrocytes in vitro. This is the first identification of antigens expressed in Babesia sp. sporozoites and establishes that, at least in part, sporozoites and merozoites share common targets of antibody mediated inhibition of erythrocyte invasion.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Vaccination-induced variation in the 140 kD merozoite surface antigen of Plasmodium knowlesi malaria.

1987 ◽  
Vol 165 (2) ◽  
pp. 359-367 ◽  
Author(s):  
F W Klotz ◽  
D E Hudson ◽  
H G Coon ◽  
L H Miller
Keyword(s):  
Monoclonal Antibodies ◽  
Malaria Infection ◽  
Rhesus Monkeys ◽  
Plasmodium Knowlesi ◽  
Rabbit Antiserum ◽  
Antigenic Determinants ◽  
Partial Protection ◽  
Erythrocyte Invasion ◽  
Merozoite Surface Antigen

Immunity to 143/140 kD schizont antigens of a monkey malaria, Plasmodium knowlesi, provides partial protection to lethal malaria infection in rhesus monkeys challenged with uncloned parasites. To determine the capacity of a cloned parasite to generate variants of the 143/140 kD antigens, immunized monkeys were challenged with a clone of P. knowlesi. Parasites recovered 8 d after inoculation with a cloned parasite retained the 143/140 kD antigens. Parasites recovered 30 d after challenge had undergone changes in the 143/140 kD antigens. Antibodies that block erythrocyte invasion in vitro of the inoculum parasites did not inhibit invasion of erythrocytes by two isolates recovered from the immunized monkeys. An isolate from one monkey recovered on day 30 contained clones expressing new 76/72 kD antigens reactive with rabbit antiserum against the 143/140 kD proteins, and other clones expressing no antigens crossreactive with antisera against the 143/140 kD proteins. An isolate from another monkey obtained 59 d after challenge expressed new antigens of 160/155, 115/113, and 87/85 kD. Using monoclonal antibodies, we found that epitopes were lost from the variant proteins, but we were unable to determine whether new epitopes had appeared. We conclude that clones of P. knowlesi can rapidly vary antigenic determinants on the 143/140 kD proteins in animals immunized with these antigens.

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