After PCR amplification using the Platinum em Pfx /em DNA polymerase enzyme (Invitrogen, California, USA), each product was purified by Wizard PCR preps kit (Promega), ligated to the pEXP5 CT/TOPO vector (Invitrogen) and cloned in em Escherichia coli /em TOP10 bacteria (Invitrogen), following the manufacturer’s instructions

After PCR amplification using the Platinum em Pfx /em DNA polymerase enzyme (Invitrogen, California, USA), each product was purified by Wizard PCR preps kit (Promega), ligated to the pEXP5 CT/TOPO vector (Invitrogen) and cloned in em Escherichia coli /em TOP10 bacteria (Invitrogen), following the manufacturer’s instructions. schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the em Pv /em RON1 protein Flt3 was assessed using human sera from individuals previously exposed to em P. vivax /em malaria by ELISA. Results In the em P. vivax /em VCG-1 strain, RON1 is usually a 764 amino acid-long protein. em In silico /em analysis has revealed that em Pv /em RON1 shares essential characteristics with different antigens involved in invasion, such as L-Thyroxine the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain name. The em Pv /em RON1 protein is expressed in parasite’s schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural contamination with em P. vivax /em . Conclusions This study shows the identification and characterization of the em P. vivax /em rhoptry neck protein 1 in the VCG-1 strain. Taking into account that em Pv /em RON1 shares several important characteristics with other em Plasmodium /em antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the L-Thyroxine em Aotus /em monkey model are thus recommended. strong class=”kwd-title” Keywords: Rhoptry, em Plasmodium vivax /em , Antigenicity, vaccine candidate Background Malaria remains one of the prevailing health problems worldwide. According to the World Health Business (WHO) [1], nearly 225 million people are infected annually; about 785,000 of them die as a direct consequence of this disease, of which 85% are children aged less than five years. Although malaria in humans is caused by em Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae /em and em Plasmodium knowlesi /em , the first two species represent about 90% of the clinical cases reported [2]. em P. falciparum /em is responsible for causing the disease’s highest mortality rates while em P. vivax /em represents significant morbidity having socioeconomic implications [3]. In spite of international control strategies and guidelines having been implemented during the last fifty years, mortality figures are still alarming; therefore, developing an efficient vaccine to combat this imminent threat has become an urgent need. Invasion of red blood cells (RBC) by em Plasmodium /em parasites involves highly coordinated events which are directed by a set of L-Thyroxine proteins secreted from the apical organelles (rhoptries and micronemes) [4]. It has been shown that several rhoptry proteins, such as em Pf /em RON2, -4, and the em Pf /em AMA-1 antigen (secreted by micronemes), are involved in tight junction formation between the parasite and its target cell [5-7]; it has also been found that some others (such as em Pf /em RON1, em Pf /em 92, em Pf /em 38, em Pf /em 12 and em L-Thyroxine Pf /em 34) are associated with detergent-resistant membrane microdomains (DRM) through glycosylphosphatidylinositol (GPI)-anchor sequences [8], which are considered organizing centers for the assembly of molecules implicated in cell signaling [9,10]. To date, several of these DRM proteins have been shown to play an active role in host cell interaction and to trigger antibody responses in the host [11-15]. Rhoptry neck protein 1 (RON1), initially described in em Toxoplasma gondii /em ( em Tg /em RON1) [16], has been a particularly interesting protein. It is a highly-conserved antigen amongst em Apicomplexa /em members. Different em L-Thyroxine tgron1 /em homologous genes have also been found in members of the em Plasmodium /em genus, such as em P. falciparum /em [16,17]. em Pf /em RON1 is also known as the apical sushi protein (ASP), exhibiting a prominent transcriptional peak towards the end of the intraerythrocyte lifecycle [17]. This protein has 731 amino acids encoded by 4 exons and ~85.46 kDa molecular mass. It’s been proven that em Pf /em ASP goes through proteolytic control previously, leading to ~50 kDa and ~30 kDa polypeptides [18]. The proteins consists of a sign peptide, a GPI-anchor series and a go with control proteins (CCP) or sushi-like conserved site [19]; this site typically consists of ~60 residues and represents a proteins module involved with protein-protein relationships and/or cell adhesion, including 4 cysteines, proline residues and extremely conserved acidic proteins (aspartic and glutamic acidity). Furthermore, it’s been recently discovered that em Pf /em RON1 offers 4 high activity binding peptide sequences (HABPs) to erythrocytes (manuscript in planning), and may make a difference for parasite admittance to focus on cells as a result. In the seek out a highly effective anti-malarial vaccine many studies have already been centered on determining antigens expressed through the parasite’s asexual stage because of the fact that a few of these antigens are primarily in charge of invasion of RBC [20,21]. Many proteins contained in developing a guaranteeing vaccine against em P. falciparum /em have already been characterized to day [21]. However, studies completed with em P. vivax /em have already been limited due.

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