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P190系恶性疟原虫裂殖子表面抗原1(MSA1),是很有希望的疟疾疫苗候选抗原。根据该基因现有序列,我们设计了3对引物,在5′端引物和3′端引物前分别设有BamHI和XbaI酶切位点序列,在每个酶切位点前设有保护性碱基GC,用固相亚磷酸酰胺法在ABI391型DNA自动合成仪上自动合成引物,合成的引物氨解、冷冻干燥后经HPLC纯化。采用聚合酶链反应(PCR)方法扩增了恶性疤原虫FCC1/HN株P190第3、4保守区和第2保守区部分序列的基因片段,分别定名为P190CR3、P190CR4和P190CR2-2,各片段均连接到pUC18载体上,用双脱氧末端终止法测定了克隆片段的DNA序列,结果表明这3个区域的DNA序列与MAD20(采自巴布亚-新几内亚)、Wellcome(采自西非)、K1(采自泰国)及CAMP(采自马尔加什)株恶性疟原虫P190相应区域比较也是高度保守的。在该基因第2保守区核苷酸第81位(相当于MAD20的732位)发生了碱基替换,由T替代了C,但未发生氨基酸的改变。
P190 Plasmodium falciparum merozoite surface antigen 1 (MSA1) is a promising malaria vaccine candidate antigen. According to the existing sequence of the gene, we designed three pairs of primers, and provided the sequences of BamHI and XbaI restriction sites before the 5 ’primer and the 3’ primer, and provided a protective base in front of each enzyme cutting site Based on GC, primers were synthesized automatically by solid-phase phosphite method on ABI391 DNA automatic synthesizer. The synthesized primers were aminolysis, freeze-dried and purified by HPLC. Polymerase chain reaction (PCR) method was used to amplify the partial sequences of the 3rd and 4th conserved regions of the 3rd and 4th conserved regions of P190 and P190, which were named as P190CR3, P190CR4 and P190CR2-2 respectively. Each fragment Were ligated to the pUC18 vector and the DNA sequence of the cloned fragment was determined by the dideoxy terminator method. The results showed that the DNA sequences of the three regions were identical to those of MAD20 (Papua New Guinea), Wellcome (West Africa), K1 From Thailand) and CAMP (from Malagasy) P. falciparum P190 corresponding region is also highly conserved. A base substitution occurred at position 81 (corresponding to position 732 of MAD20) in nucleotide 2 of the conserved region of the gene, and C was replaced by T, but no amino acid change was observed.