Genes for Plasmodium falciparum surface antigens cloned by expression in COS cells.

Abstract

Two genes encoding membrane antigens of Plasmodiumfalciparum were isolated by transient expression in mammalian cells and selection with human immune sera from African adults exposed to P. falciparum malaria. COS-7 cells were transfected with a plasmid expression library constructed from P. falciparum genomic DNA, and cells expressing reactive malaria antigens on their surface were enriched by adherence to antibody-coated dishes. One of the genes isolated is distinctive in that it does not contain repeat sequences typical of many malarial genes cloned by immunoscreening of bacterial expression libraries. The second gene apparently encodes a polymorphic version of the P. falciparum merozoite surface antigen Ag513, since the two sequences are identical in the 5' and 3' coding regions but diverge completely in the center. The COS-7 expression system provides an alternate means for cloning genes encoding malarial membrane antigens by using those antibodies in complex immune sera that bind membraneassociated, nondenatured molecules.Proteins expressed on the surface of parasites constitute potential target antigens for vaccine development. These molecules are accessible to the immune system and also may mediate functions vital for parasite survival such as cell attachment, host-cell invasion, or membrane transport. In the case of Plasmodium falciparum, surface membrane antigens of the asexual blood stages are of special interest because these stages are responsible for the high morbidity and mortality of P. falciparum malaria. The P. falciparum genes characterized to date have been cloned largely in bacterial expression systems, typically by using Agtll cDNA or genomic libraries in Escherichia coli and screening with human immune serum (1, 2). However, with these methods few genes for malarial surface antigens have been cloned, possibly due to poor expression of membrane proteins in E. coli and the fact that bacterial fusion proteins lack epitopes dependent on native secondary or higher order protein conformations. In this paper we show that genes encoding parasite membrane proteins can be isolated directly by expression in mammalian cells and selection for cells bearing heterologous surface antigens by using human immune serum from individuals exposed to the infection of interest. This method should be generally applicable to a number of parasitic diseases. Although the genes for a number of different human cell surface antigens have been cloned by transient expression in mammalian cells, these have been isolated exclusively from cDNA libraries, and immunological selection has depended on specific monoclonal antibodies (3-6). We demonstrate that genomic libraries can also be used in similar expression cloning and that polyspecific antibodies from human serum can be used for selection of transfected cells. MATERIALS AND METHODScDNA Expression Vector pJFE14. This simian virus 40 (SV40)-based COS cell expression vector is derived from pSSD2. The vector pSSD2 is similar to the original Okayama and Berg vector pcD...

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution

“…Expression Constructs and Generation of Chimeras All cDNAs and chimeras were expressed in COS-7 cells by using the SR-u-based expression vector pJEF14 (Elliott et al, 1990), modified as described in Tsim et al (1992). All DNA constructs were subcloned into this vector using the EcoRI site in its polylinker.…”

Structure of thePlasmodium6-cysteine s48/45 domain

“…Pf12, Pf38, and Pf41 are readily recognized by immune sera from infected patients from endemic areas (10,25,26); however, no function has been ascribed to any of them yet. The first step in invasion of merozoites into red blood cells is the binding of any part of the merozoite to the red blood cell surface.…”

“…Because Pf41 has no GPI anchor signal or transmembrane domain for attachment to the membrane, it is likely to bind another merozoite surface protein. Pf41, Pf38, and Pf12 are strongly recognized by immune sera from naturally infected patients, and Pf38 and Pf92 are under balancing selection (10,(25)(26)(27). Attempts to delete Pf92 have been reportedly unsuccessful; however, no information for the deletion of the other genes is available (28).…”

Activation loop tyrosines contribute varying roles to TrkB autophosphorylation and signal transduction

“…Recombinant human BDNF and the pBJ5 plasmid (Elliot et al, 1990) were kind gifts from Andy Welcher, AMGEN (Thousand Oaks, CA), Phospho-speci®c ERK1/2 antibody was purchased from New England Biolabs (Beverly, MA). Anti-ERK1/2 (SC-4) and anti-c-fos antibody (SC-52) were both purchased from Santa Cruz Labs (Santa Cruz, CA).…”