title: Molecular characterization of calreticulin from Anopheles stephensi midgut cells and functional assay of the recombinant calreticulin with Plasmodium berghei ookinetes.
abstract: Transmission blocking vaccines (TBVs) that target the antigens on the midgut epithelium of Anopheles mosquitoes are among the promising tools for the elimination of the malaria parasite. Characterization and analysis of effective antigens is the first step to design TBVs. Calreticulin (CRT), a lectin-like protein, from Anopheles albimanus midgut, has shown antigenic features, suggesting a promising and novel TBV target. CRT is a highly conserved protein with similar features in vertebrates and invertebrates including anopheline. We cloned the full-length crt gene from malaria vector, Anopheles stephensi (AsCrt) and explored the interaction of recombinant AsCrt protein, expressed in a prokaryotic system (pGEX-6p-1), with surface proteins of Plasmodium berghei ookinetes by immunofluorescence assay. The cellular localization of AsCrt was determined using the baculovirus expression system. Sequence analysis of the whole cDNA of AsCrt revealed that AsCrt contains an ORF of 1221 bp. The amino acid sequence of AsCrt protein obtained in this study showed 64% homology with similar protein in human. The AsCrt shares the most common features of CRTs from other species. This gene encodes a 406 amino-acid protein with a molecular mass of 46 kDa, which contains a predicted 16 amino-acid signal peptides, conserved cysteine residues, a proline-rich region, and highly acidic C-terminal domain with endoplasmic reticulum retrieval sequence HDEL. The production of GST-AsCrt recombinant protein was confirmed by Western blot analysis using an antibody against the GST protein. The FITC-labeled GST-AsCrt exhibited a significant interaction with P. berghei ookinete surface proteins. Purified recombinant GST-AsCrt, labeled with FITC, displayed specific binding to the surface of P. berghei ookinetes in comparison with control. Moreover, the expression of AsCrt in baculovirus expression system indicated that AsCrt was localized on the surface of Sf9 cells. Our results suggest that AsCrt could be utilized as a potential target for future studies in TBV area for malaria control.
authors.affiliation: Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran; Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Nahid
authors.initials: N
authors.lastname: Borhani Dizaji
authors.affiliation: Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Basseri
authors.affiliation: Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Saied Reza
authors.initials: SR
authors.lastname: Naddaf
authors.affiliation: Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran; Stem Cell Preparation Unit, Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran. Electronic address: mheidari@sina.tums.ac.ir.
authors.forename: Mansour
authors.initials: M
authors.lastname: Heidari
mesh_terms: D000818:Animals; D000852:Anopheles / Q000235:genetics* / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D037282:Calreticulin / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018014:Gene Transfer Techniques; D007303:Insect Vectors; D007413:Intestinal Mucosa / Q000378:metabolism*; D051379:Mice; D033661:Oocysts / Q000378:metabolism; D030781:Organisms, Genetically Modified; D010962:Plasmodium berghei / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D037282:Calreticulin; D011994:Recombinant Proteins
keywords: Anopheles stephensi; Calreticulin; Interaction assay; Localization; Molecular analysis; Plasmodium berghei; Protein characterization; TBV