Pf332抗原的结构和抗原表位的初步研究

目的　了解恶性疟原虫FCCl／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FCCl／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9595－10083、10339－10767和10855－11247位碱基对应的RO、R1和R2片段分别插入真核表达载体pcDNA3-S。将pcDNA3-R0、pcDNA3-S-R1和pcDNA3-S-R2分别免疫Balh/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16377bp，编码5458个氨基酸，相对分子质量约615.28ku。Ag332包含17个调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FCCl/HN株和3D7株Ag332的氨基酸残基同源性达94.55%。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,pcDNA3-S-R1和pcDNA3-S-R2的实验组IgG含量显大于空白对照组和pcDNA3组（P＜0.05)。体外疟原虫生长抑制实验结果表明，pcDNA3-S-R0,pcDNA3-S-RI和pcDNA3-S-R2组的免疫血清在1：5稀释度时对恶性疟原虫的生长有抑制作用。结论　Pf332抗原是一包含很多调度简并的富谷氨酸重复序列的大蛋白，Pf332基因片段R1和R2编码潜在的抗原表位重复序列。

Primary Study of the Structure and Antigenic Epitopes of Antigen Pf332

Objective To understand the primary structure and potential antigenic epitopes of antigen Pf332(Ag332) of P.falciparum iso late FCC1/HN.Methods　Based on the published Pf332 gene sequence , nine pairs of primers were designed for the PCR amplification of the Pf332 gen e fragments from genomic DNA of P.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, cor responding to nt#9595-10083, nt#10339-10767 and nt#10855-11247 of Pf332 gene were subcloned into the eukaryotic expression vector pcDNA3-S separately. The Balb/c mice were immunized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3- S-R2 separately, and the expressions of the recombinant proteins were detected by immunohistochemistry assay. The protective immune responses elicited by DNA I mmunization were analyzed by ELISA and parasite growth inhibition tests in vitro .Results　Nine Pf332 gene fragments were specifically amplif ied, subcloned into pMD-18T vectors and sequenced. Pf332 gene of the P.falci parum isolate FCC1/HN was 16,377 bp in length, encoding a protein of 5,458 ami no 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 of P.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 immu nized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 were higher than those of blank and pcDNA3 groups (P＜0.05). The result of parasite growth inhibition test showed that the immunized sera at 1∶5 dilution of groups of pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 had an incomplete inhibitor y effect on P.falciparum growth. Conclusion　The antigen Pf332 is an large protein containing highly degenerated Glu-rich repeats. Pf332 gene fragments, R1 and R2 encoding potent antigenic epitope repeats.