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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 ,为以后其结构与功能的研究奠定基础
Objective To clone the 175 ku erythrocyte binding antigen (EBA-175) gene of Plasmodium falciparum FCC1 / HN strain into sequencing vector and determine its sequence, which will lay the foundation for further study of its structure and function. Methods The full-length coding sequence of EBA-175 gene was amplified from the genomic DNA of Plasmodium falciparum FCC1 / HN strain in two fragments by PCR amplification. After purification and recovery of the amplified product, the T-A gene was cloned into the sequencing vector pMD18-T and transformed into E. coli DH5α. The positive clones were screened and double-digested with restriction endonucleases (PCR) - 175 gene recombinant plasmid p MD18-T-EBA. DNA sequencing was performed using the Sanger dideoxy chain termination method. Results The sequence of EBA-175 gene of FCC1 / HN strain was almost the same as that of Camp strain. It contained 4308bp in length and encoded 1435 amino acids, which contained a C fragment similar to Camp strain. Antigenic epitope analysis of F2 sub-region and 4-peptide of RII region using computer software revealed that these regions may contain epitopes. Conclusion The determination of EBA-175 whole-genome coding sequence laid the foundation for its future study on its structure and function