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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 ,为获得Pf332全基因
To determine the unknown sequence of the Pf332 antigen (Ag332) of Plasmodium falciparum in the red stage and to carry out sequence analysis.A pair of primers was designed according to the G1 fragment of the Pf332 gene of P. falciparum Palo alto strain, FCC1 HN) genomic DNA was amplified P332 1 fragment.Pf332 gene often SVTEEI short peptide coding sequence, respectively, based on the design of non-specific sense of antisense oligonucleotide primers (NSP1, NSP2), the application of low stringency PCR (LSPCR) were used to amplify the P332 up1 and P332 dow1 fragments adjacent to P332 1 respectively.According to the sequence of G9 and C1 fragments upstream and downstream of G1 fragment of Pf332 gene of P. falciparum and the P332 up1 and P332 dow1 sequences , Two pairs of specific primers were designed to continue to amplify the adjacent unknown sequence fragments P332 up2 and P332 dow2.According to the 3 ’end sequence of P332 dow2 fragment, two specific primers were designed respectively with non-specific primer NSP2 for LSPCR and nested PCR P332 dow3 adjacent to unknown sequence fragment P332 Dow3. Pf332 gene fragment obtained sequence determination, and molecular biology software assisted sequence analysis. The sequencing and splicing results showed that a total of 614 bp of Pf332 gene sequence of P. falciparum FCC1 HN strain was obtained.Sequence analysis showed that the 614 bp sequence was located in the coding region of Pf332 gene, 0 48 amino acid residues, including five amino acid residue repeats.Pf332 gene of Plasmodium falciparum FCC1 HN strain 6 14 4bp sequence determination and analysis, in order to obtain Pf332 whole gene