恶性疟原虫红内期Pf332抗原基因未知序列的测定及分析

Sequence Determination and Analysis of Unknown Fragments of Blood Stage Antigen Pf332 Gene of Plasmodium falciparum

测定恶性疟原虫红内期Pf332抗原 (Ag332 )基因的未知序列 ,并进行序列分析 .根据非洲恶性疟原虫Palo alto株Pf332基因的G1片段序列 ,设计 1对引物 ,从中国恶性疟原虫海南株 (FCC1 HN)基因组DNA中扩增出P332 1片段 .Pf332基因中经常出现SVTEEI短肽的编码序列 ,据此分别设计非特异的正、反义寡核苷酸引物 (NSP1、NSP2 ) ,应用低严谨PCR(LSPCR)分别扩增出P332 1邻近的未知序列片段P332 up1和P332 dow1.根据恶性疟原虫Palo alto株Pf332基因G1片段上、下游的G9和C1片段序列以及测定的P332 up1和P332 dow1序列 ,分别设计 2对特异引物继续扩增邻近的未知序列片段P332 up2和P332 dow2 .根据P332 dow2片段的 3'端序列 ,设计 2条特异引物分别与非特异引物NSP2行LSPCR和巢式PCR ,扩增出P332 dow2邻近的未知序列片段P332 dow3.对获得的Pf332基因片段进行序列测定 ,并用分子生物学软件辅助进行序列分析 .序列测定和拼接结果显示 ,共获得了连续 6 14 4bp的恶性疟原虫FCC1 HN株Pf332基因序列 .序列分析表明 ,所获得的 614 4bp序列位于Pf332基因的编码区内 ,不含内含子 ,编码 2 0 4 8个氨基酸残基 ,包含 5个氨基酸残基重复区 .对恶性疟原虫FCC1 HN株Pf332基因 6 14 4bp序列的测定和分析 。

To determine and analyze the unknown sequence of the blood stage antigen Pf332(Ag332) gene of \%Plasmodium falciparum\%, a pair of primers were designed, synthesized and used to amplify p332 1 from the genomic DNA of \%P.falciparum\% strain FCC1/HN according to the published sequence of fragment G1 of Pf332 gene of \%P.falciparum\% stain Palo\|alto.Since the nucleotides encoding SVTEEI disperse frequently in the known Pf332 gene fragments, a sense and an antisense nonspecific primers, NSP1 and NSP2, corresponding to the nucleotides encoding SVTEEI were designed. Then using low stringency PCR(LSPCR), the unknown fragments , P332 up1 and P332 dow1, overlapped with P332 1 were amplified. According to the sequences of the upstream fragment G9 and the downstream fragment C1 of Pf332 gene fragment G1 of \%P.falciparum\% strain Palo alto, and the sequence of P332 up1 and P332 dow1, two pairs of primers were synthesized and used to amplify the unknown fragments P332 up2 and P332 dow2,separately. According to the sequences of the 3'region of P332 dow2, two specific primers were synthesized and used to amplify the unknown fragment P332 dow3 overlapped with P332 dow2 by LSPCR and nested PCR with primer NSP2. The amplified Pf332 gene fragments were sequenced, and the obtained Pf332 gene sequence was analyzed by using molecular biology software. The result of sequence determination and conjunction shows that a linear sequence about 6 144 bp of Pf332 gene of \%P.falciparum\% strain FCC1/HN was obtained. Sequence analysis showed that 6 144 bp sequence within the open reading frame (ORF) of Pf332 gene, with no introns, encodes 2 048 amino acid residues , including 5 regions of amino acid repeats. The sequence determination and analysis of the 6 144 bp fragment of Pf332 gene of \%P.falciparum\% strain FCC1/HN lay a foundation for sequencing the full length Pf332 gene and further study on structure and function of Pf332 gene.