恶性疟原虫FCC1/HN株EBA-175基因克隆及序列分析

CLONING AND SEQUENCING OF THE GENE ENCODING THE 175-ku ERYTHROCYTE BINDING ANTIGEN OF PLASMODIUM FALCIPARUM ISOLATE FCC1/HN

目的 将恶性疟原虫 FCC1/ HN株 175 ku的红细胞结合抗原 (EBA- 175 )基因克隆入测序载体 ,测定其序列 ,为以后研究其结构与功能奠定基础。 方法 利用 PCR扩增技术 ,分两个片段从恶性疟原虫 FCC1/ HN株基因组 DNA中 ,特异扩增 EBA- 175全基因编码序列。扩增产物经纯化回收后 ,T- A克隆入测序载体 p MD18- T,转化大肠杆菌 (E.coli) DH5 α,筛选阳性克隆 ,并进行双酶切及 PCR扩增鉴定 ,获得含有编码 EBA- 175基因的重组质粒 p MD18- T- EBA。用Sanger双脱氧链终止法进行 DNA序列测定。 结果 FCC1/ HN株 EBA- 175基因序列与 Camp株基本一致 ,全长 430 8bp,编码 1435个氨基酸 ,含有与 Camp株相似的 C片段。利用计算机软件对其 RII区的 F2亚区以及 4肽进行抗原表位分析 ,结果显示这些区域可能含有抗原表位。 结论 EBA- 175全基因编码序列的测定 。

Objective Sequence the 175 ku erythrocyte binding antigen (EBA-175) of Plasmodium falciparum isolate FCC1/HN for its structural and functional studies. Methods According to the published nucleotide sequence of the EBA-175 of P. falciparum Camp strain, two pairs of oligonucleotides were designed as primers(P1,P2 and P3,P4). Using polymerase chain reaction (PCR) technique, the gene of EBA-175 was specifically amplified from the genomic DNA of P. falciparum isolate FCC1/HN. The PCR products were puried and cloned into plasmid pMD18-T by T-A cloning method, transformed into Escherichia coli(E. coli) strain DH5α. The recombinant plasmids were screened and identified by BamHI and SalI digestion and PCR amplification. Results DNA sequencing demonstrated a single open reading frame encoding a translation product of 1 435 amino-acid residues. The predicted protein sequence was highly conserved with that predicted from of the EBA-175 gene sequences from Camp strain and contained the C segment divergent region. There also may be antigenic epitopes in RII(F2) and EBA-piptide 4. Conclusion These finding are important for the structural and functional studies of the EBA-175 gene.