High throughput immuno-screening of cDNA expression libraries produced by in vitro recombination; exploring the Plasmodium falciparum proteome.
Improved Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo-capping with in vitro recombination and directional cloning of cDNA inserts into a plasmid vector that expresses sequences as thioredoxin fusion proteins. A novel procedure has also been developed for the rapid identification of seropositive clones on high-density filters, using direct labelling of P. falciparum immune immunoglobulin with fluorescein isothiocynate (FITC). This approach combines the advantages of recombination-assisted cDNA cloning with high throughput, non-radioactive serological screening of expression libraries. Production of replicate colony matrices allows the identification of antigens recognised by different pools with different specificities from residents of a malaria endemic region. Analyses of DNA sequences derived from sero-reactive colonies indicate that this is an effective method for producing recombinant proteins that react with antibodies from malaria-exposed individuals. This approach permits the systematic construction of a database of antigenic proteins recognised by sera from malaria-exposed individuals.
Sowa
M P Kordai
MP
Institute of Cell, Animal and Population Biology, Ashworth Laboratories, Kings' Buildings, University of Edinburgh, West Mains Road, Edinburgh, Scotland, UK. kordai@holyrood.ed.ac.uk
Sharling
Lisa
L
Humphreys
Georgina
G
Cavanagh
David R
DR
Gregory
William F
WF
Fenn
Katelyn
K
Creasey
Alison M
AM
Arnot
David E
DE
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood / Q000276:immunology; D000953:Antigens, Protozoan / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D010963:Plasmodium falciparum / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D015800:Protozoan Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D011995:Recombination, Genetic; D017385:Sequence Homology
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D020543:Proteome; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins