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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?
Objective To determine the sequence of the Ameloblastoma antigen 1 (AMA - 1) gene and the Pfs2 30 gene of the Plasmodium falciparum Hainan (FCC1 / HN) strain and to analyze the sequence separately. Methods A pair of primers was synthesized based on the known sequence of AMA - 1 gene. The AMA - 1 gene was amplified from genomic DNA of Plasmodium falciparum FCC1 / HN by PCR and the eukaryotic recombinant plasmid pcDNA3 - AMA - 1 was constructed. Seven pairs of primers were synthesized based on the known sequence of Pfs230 gene. The Pfs230 gene was amplified from genomic DNA of FCC1 / HN strain in seven sections and inserted into the pMD - 18T sequencing vector respectively. The sequences of the cloned AMA-1 and Pfs230 genes were determined by the dideoxy chain termination method, and the sequence analysis and homology comparison were assisted by DNAstar software. Results The AMA - 1 and Pfs2 30 gene fragments of Plasmodium falciparum FCC1 / HN strain were amplified by PCR. The AMA - 1 gene of Plasmodium falciparum FCC1 / HN strain was 186 9 bp in length and contained no intron, which encoded 6222 amino acid residues without any amino acid repeats and had a relative molecular mass of about 72 0 4 5 kDa. The Pfs2 30 gene was 94 35bp, no intron, encoding 314 4 amino acid residues, the molecular weight of 36 4 36kDa. The homology of Plasmodium falciparum FCC1 / HN strains to FC2 7,7G8, CAMP, FCR3, Thai -Tn, 3D7, FVO, KF1916, CMP1, HB3, K1 and V1 strain AMA- 1 was above 94 9% A total of 53 alternative amino acid residue substitution sites were found, and alternative amino acid residues were found to be binary. The FCC1 / HN strain has 9 and 10 amino acid residues more than the P730 strain of the 3D7 and 7G8 strains, respectively, and the two isolates have 28 amino acid substitutions?