[Cloning and sequencing of the gene coding the sexual stage antigen Pfs48/45 of Plasmodium falciparum].
OBJECTIVE
To express the antigen Pfs48/45 in vitro and provide an antigen for the development of the transmission-blocking vaccine.


METHODS
According to the published nucleotide sequence of Pfs48/45 of Plasmodium falciparum isolate NF54, a pair of oligonucleotides was designed and used as primers(P1, P2). The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of P. falciparum isolate FCC1/HN has been amplified by using polymerase chain reaction (PCR) technique. The PCR product was purified and directly sequenced by the dideoxynucleotide terminator method with 5'-end primer P1. At the same time, the purified PCR product was digested with BamHI and EcoRI and cloned into the plasmid pcDNA3, then the recombinant clones were transformed into E. coli strain TG1. The recombinant plasmid pcDNA3-Pfs48/45 was screened and identified by PCR amplification and restriction analysis.


RESULTS
1. The gene fragment Pfs48/45 was specifically amplified from the genomic DNA of Plasmodium falciparum isolate FCC1/HN; 2. The sequence demonstrated that the that the 5'-end nucleotide and predicted aminoacid sequence of Pfs48/45 from FCC1/HN isolate was basically identical with that from NF54 isolate. We found that the sequence of the Pfs48/45 gene from the isolate FCC1/HN differs from the published sequence(isolate NF54) only at positions 307 and 372(T-->C). The substitution of T-->C at the position of 372 generates a new restriction site Taq I. The PCR product digested by Taq I generates DNA fragment of 984 bp and 379 bp, suggesting that the PCR product is the gene encoding the transmission blocking antigen Pfs48/45; 3. The gene fragment of Pfs48/45 was directly inserted into the BamHI and EcoRI site of plasmid pcDNA3.


CONCLUSIONS
The nucleotide sequence of Pfs48/45 of Plasmodium falciparum isolate FCC1/HN from south China was similar to that of isolate NF54. The recombinant plasmid pcDNA3-Pfs48/45 was successfully constructed, providing a means to evaluate the role and biological function of this sexual-stage-specific protein of Pfs48/45.
Luo
S
S
Department of Parasitology, Sun Yat-sen University of Medical Sciences, Guangzhou 510089.
Yu
X
X
Liu
Y
Y
Fang
J
J
Xu
J
J
Li
X
X
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics*; D017125:Genes, Protozoan; D017780:Malaria Vaccines; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000276:immunology*; D015800:Protozoan Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA
D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D017780:Malaria Vaccines; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; C084847:pfs48-45 protein, Plasmodium falciparum