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目的 了解恶性疟原虫FCC1/HN株Pf332抗原 (Ag332 )的初级结构和潜在的抗原表位。方法 根据Pf332基因已知序列 ,合成 9对引物用于从恶性疟原虫FCC1/HN株基因组DNA中扩增Pf332基因片段。将扩增得到的 9个Pf332基因片段插入pMD 18T载体后测序。用DNAstar软件将测定的Pf332基因片段序列拼接出成完整的Pf332基因序列。分别用SAPS、Tmpred、SingalP和Blastn程序来分析Ag332的初级结构和序列同源性。将与Pf332基因的 95 95～ 10 0 83、10 339～ 10 76 7和 10 85 5～ 112 4 7位碱基对应的R0、R1和R2片段分别插入真核表达载体pcDNA3 S。将pcDNA3 S R0、pcDNA3 S R1和pcDNA3 S R2分别免疫Balb/c鼠 ,并通过免疫组化检测表达产物。通过ELISA和体外疟原虫生长抑制实验来鉴定DNA免疫所诱导的保护性免疫反应。结果 扩增到特异的 9个Pf332基因片段 ,并正确插入pMD 18T载体。序列测定和拼接结果显示 ,恶性疟原虫FCC1/HN株Pf332基因长 16 377bp ,编码 5 4 5 8个氨基酸 ,相对分子质量约 6 15 2 8ku。Ag332包含 17个高度简并的富谷氨酸重复序列 ,抗原中谷氨酸占 30 18%。恶性疟原虫FCC1/HN株和 3D7株Ag332的氨基酸残基同源性达 94 5 5 %。免疫组化检测显示R0、R1和R2在鼠肌肉组织中表达。分别免疫了pcDNA3 S R0、
Objective To understand the primary structure and potential epitope of Pf332 antigen (Ag332) of Plasmodium falciparum FCC1 / HN strain. Methods According to the known sequence of Pf332 gene, 9 pairs of primers were designed to amplify the Pf332 gene from genomic DNA of Plasmodium falciparum FCC1 / HN strain. The amplified 9 Pf332 gene fragments were inserted into pMD 18T vector and sequenced. The Pf332 gene fragment was sequenced by DNAstar software to complete the Pf332 gene sequence. The primary structure and sequence homology of Ag332 were analyzed using SAPS, Tmpred, SingalP and Blastn programs, respectively. The R0, R1 and R2 fragments corresponding to the bases 95 95-103, 10 339-1076 7 and 10 855-112 4 7 of the Pf332 gene were inserted into the eukaryotic expression vector pcDNA3-S. Balb / c mice were immunized with pcDNA3 S R0, pcDNA3 S R1 and pcDNA3 S R2 respectively, and the expression products were detected by immunohistochemistry. Protective immunity induced by DNA immunization was identified by ELISA and in vitro growth inhibition assays of Plasmodium. As a result, 9 specific Pf332 gene fragments were amplified and correctly inserted into the pMD 18T vector. Sequence analysis and splicing showed that the Pf332 gene of P. falciparum FCC1 / HN strain was 16 377 bp in length and encoded a total of 5458 amino acids with a relative molecular mass of 6 15 2 8 ku. Ag332 contains 17 highly degenerate glutamate-rich repeats, with 30 18% of the glutamate in the antigen. The amino acid residues of P. falciparum FCC1 / HN strain and 3D7 strain Ag332 were 94.55%. Immunohistochemistry showed that R0, R1 and R2 were expressed in murine muscle tissue. Were immunized with pcDNA3 S R0,