恶性疟原虫海南株AMA-1、Pfs230基因的序列分析

Sequence analysis of AMA-1 and Pfs230 gene of Plasmodium falciparum isolate FCC1/HN

目的 测定恶性疟原虫海南 (FCC1/HN)株裂殖子顶端膜抗原 1(AMA - 1)基因和Pfs2 30基因序列 ,并分别进行序列分析。方法 根据AMA - 1基因已知序列合成一对引物 ,用PCR技术从恶性疟原虫FCC1/HN株基因组DNA中扩增AMA - 1基因 ,构建真核表达重组质粒 pcDNA3 -AMA - 1。根据Pfs2 30基因已知序列合成七对引物 ,分 7段从FCC1/HN株基因组DNA中扩增Pfs2 30基因 ,并分别将扩增片段插入pMD - 18T测序载体。用双脱氧链末端终止法测定克隆的AMA -1、Pfs2 30基因序列 ,应用DNAstar软件辅助进行序列分析和同源性比较。结果 PCR扩增得到恶性疟原虫FCC1/HN株AMA - 1和Pfs2 30基因片段。恶性疟原虫FCC1/HN株AMA - 1基因全长 186 9bp ,无内含子 ,编码 6 2 2个氨基酸残基 ,不存在氨基酸重复序列 ,相对分子量约 72 0 4 5kDa ;Pfs2 30基因全长 94 35bp ,无内含子 ,编码 314 4个氨基酸残基 ,分子量为36 4 36kDa。恶性疟原虫FCC1/HN株与FC2 7、7G8、CAMP、FCR3、Thai -Tn、3D7、FVO、KF1916、CMP1、HB3、K1和V1株AMA - 1的同源性在 94 9%以上 ,各株间有 5 3个位置相同的氨基酸残基替代位点 ,并且发生替代的氨基酸残基具二态性。FCC1/HN株分别比 3D7、7G8株Pfs2 30抗原多 9、10个氨基酸残基 ,三个分离株有 2 8个氨基酸替换?

Aim To determine the nucleotide sequence of the apical membrane antigen 1(AMA 1) gene and the Pfs230 gene of Plasmodium falciparum isolate FCC1/HN,and to analyze the differences of sequences of AMA 1 and Pfs230 of P falciparum isolate FCC1/HN and other ones Methods According to the known sequence of AMA 1 gene,a pair of primers was designed and used to amplify the AMA 1 gene from the genomic DNA of P falciparum isolate FCC1/HN,and the AMA 1 gene was inserted into pcDNA3 vector According to the known sequence of Pfs230 gene,seven pairs of primers were designed and used to amplify Pfs230 gene fragments overlapped from genomic DNA of P falciparum isolate FCC1/HN Then the Pfs230 gene fragments were inserted into pMD18 T vectors by T A cloning method The nucleotide sequence of the AMA 1 and Pfs230 gene were determined by the dideoxy chain termination method The DNAstar software was used to analyze the sequence homology of AMA 1 and Pfs230 genes among different P falciparum isolates Results The AMA 1 and Pfs230 gene fragments of isolate FCC1/HN were specifically amplified The result of nucleotides sequencing showed that the AMA 1 gene of P falciparum isolate FCC1/HN was 1 869 base pairs in full length,without introns,encoding 622 amino acid residues,about 72 045 kDa in molecular weight,without amino acid repeats The Pfs230 gene of isolate FCC1/HN was 9 435 base pairs in length,with no introns,encoding 3 144 amino acids,about 364 36 kDa in weight The AMA 1 of P falciparum isolate FCC1/HN,FC27,7G8,CAMP,FCR3,Thai Tn,3D7,FVO,KF1916,CMP1,HB3,K1 and V1 exhibited high homology over 94 9 %,with 53 dimorphous amino acid substitutions Compared with P falciparum isolates FCC1/HN,there were 9,10 amino acid deletions in isolate 3D7 and 7G8 respectively,with 28 amino acid substitutions in Pfs230 antigens of the three isolates Conclusion Sequence determination and analysis showed that the AMA 1 and Pfs230 of different P falciparum isolates shared quite high homology respectively