CONSTRUCTION OF EUKARYOTIC EXPRESSION PLASMID AND SEQUENCE ANALYSIS OF PARTIAL Pf332 GENE OF PLASMODIUM FALCIPARUM ISOLATE FCC1/HN

Aim To construct a eukaryotic expression plasmid contained a partial gene encoding erythrocyte membrane-associated giant protein (Pf332) of Plasmodium falciparum isolate FCC1/HN,determine the sequence of Pf332 gene and compare with the sequences of Pf332 genes of isolate 3D7 and Palo Alto.Methods A pair of primers were designed based on the known sequence of Pf332 gene.Using PCR technique,Pf332 gene was obtained by amplification from genomic DNA of isolate FCC1/HN.By cloning target gene into a eukaryotic expression vector,pcDNA 3,a recombinant plasmid pcDNA 3 -Pf332 was constructed and transferred into E.coli DH5α.The positive recombinant pcDNA 3 -Pf332 was screened and identified by agarose gel electrophoresis,endonuclease digestion and PCR technique.The correct recombinant plasmid was used as template,and the nucleotide sequence of the Pf332 gene was determined by the dideoxy chain termination method.Using softwares to analyze the sequence of partial Pf332 gene and the gene homology of isolate FCC1/HN,3D7 and Palo Alto.Results The partial Pf332 gene of isolate FCC1/HN was specifically amplified,and the correct recombinant plasmid pcDNA 3 -Pf332 was constructed.The result of sequencing the nucleotide acids showed that the partial Pf332 gene of isolate FCC1/HN was 1 260 base pairs,encoding 420 amino acid residues.There were separately 1 and 15 different amino acid residues encoded by partial Pf332 genes of isolate FCC1/HN and 3D7,isolate FCC1/HN and Palo Alto.Conclusion The partial gene encoding Pf332 was amplified from genomic DNA of Plasmodium falciparum isolate FCC1/HN.The pcDNA 3 -Pf332 recombinant was successfully constructed and nucleotide sequence of Pf332 gene of isolate FCC1/HN was determined.The homology of amino acid residues encoded by partial Pf332 genes of isolate FCC1/HN and 3D7 was higher than that of isolate FCC1/HN and Palo Alto.