Pf332抗原的结构和抗原表位的初步研究(英文)  

Primary Study of the Structure and Antigenic Epitopes of Antigen Pf332

作　　者：单志新[1,2] 余新炳[1,2] 徐劲[1,2] 吴忠道[1,2] 李学荣[1,2] 卞国武[1,2] 马长玲[1,2] 李焱[1,2] 陈守义[1,2] 胡旭初[1,2] 

机构地区：[1]中山大学中山医学院病原生物学教研室 [2]广州510089

出　　处：《热带医学杂志》2002年第3期219-224,共6页Journal of Tropical Medicine

摘　　要：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 antigenic epitopes of antigen Pf332(Ag332) ofP.falciparum isolate FCC1/HN.Methods Based on the published Pf332 gene sequence, nine pairs of primers were designed for the PCR amplification of the Pf332 gene fragments from genomic DNA ofP.falciparum isolate FCC1/HN. The amplified gene fragments were subcloned into pMD-18T vectors and sequenced. The sequences were aligned using DNAstar software to obtain the full-length sequence of the gene Pf332. The primary structure and sequence homology of Ag332 were analyzed by SAPS, Tmpred, SingalP and Blastn programs. Three fragments, R0, R1 and R2, corresponding to nt#9595-10083, nt#10339-10767 and nt#10855-11247 of Pf332 gene were subcloned into the eukaryotic expression vector pcDNA 3-S separately. The Balb/c mice were immunized with pcDNA 3-S-R0, pcDNA 3-S-R1 and pcDNA 3-S-R2 separately, and the expressions of the recombinant proteins were detected by immunohistochemistry assay. The protective immune responses elicited by DNA immunization were analyzed by ELISA and parasite growth inhibition tests in vitro.Results Nine Pf332 gene fragments were specifically amplified, subcloned into pMD-18T vectors and sequenced. Pf332 gene of theP.falciparum isolate FCC1/HN was 16,377 bp in length, encoding a protein of 5,458 amino acids, about 615 28kDa. The Ag332 contains 17 regions of highly degenerated Glu-rich repeats, with 30 18% Glu in total amino acids of Ag332. Ag332 ofP.falciparum isolate FCC1/HN and 3D7 exhibited 94 55 % homology in amino acid residues. The results of immunohischemistry assay showed that R0, R1 and R2 were expressed in mice muscle tissue. The amount of IgG antibody of the groups immunized with pcDNA 3-S-R0, pcDNA 3-S-R1 and pcDNA 3-S-R2 were higher than those of blank and pcDNA 3 groups (P<0 05). The result of parasite growth inhibition test showed that the immunized sera at 1∶5 dilution of groups of pcDNA 3-S-R0, pcDNA 3-S-R1 and pcDNA 3-S-R2 had an incomplete inhibitory effect on

关 键 词：恶性疟原虫 Pf32抗原 抗原表位 免疫组化检测 保护性免疫反应 

分 类 号：R382.31[医药卫生—医学寄生虫学]