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【目的】克隆并测定恶性疟原虫海南株 (FCC1/HN)肝期抗原 1基因 (LSA 1) 3′端序列 ,比较FCC1/HN株与国外分离株LSA 13′端序列的差异。【方法】应用PCR技术从FCC1/HN株基因组DNA中扩增LSA 13′端序列 ,用T A克隆法将其克隆入pMD18 T载体。阳性克隆的重组质粒经酶切及PCR扩增鉴定后 ,用双脱氧链末端终止法进行基因序列测定。应用DNASTAR软件进行不同分离株LSA 13′端序列的同源性分析。【结果】PCR扩增得到特异的FCC1/HNLSA 13′端序列。酶切及PCR鉴定获得了正确的 pT LSA 1重组质粒。测序表明 ,所克隆的LSA 13′端大小为 795bp ,编码 2 6 4个氨基酸。序列分析表明 ,我国恶性疟原虫FCC1/HN株与国外的NF5 4、T9/ 96、KEN、PNG及BRA株LSA 13′端编码的氨基酸序列只在 3个位点存在不同。【结论】克隆了恶性疟原虫FCC1/HNLSA 13′端片段。序列测定及同源性分析表明 ,FCC1/HNLSA 13′端序列与其它分离株有高度的同源性。
【Objective】 The purpose of this study was to clone and determine the 3 ’end sequence of the LSA 1 gene of the Plasmodium falciparum Hainan (FCC1 / HN) and to compare the difference of the 13’ end sequence between the FCC1 / HN strain and the foreign isolate LSA1. 【Method】 PCR was used to amplify the 13 ’end of LSA from the genomic DNA of FCC1 / HN strain and cloned into pMD18 T vector by TA cloning method. The recombinant plasmids of positive clones were identified by restriction enzyme digestion and PCR amplification. The gene sequences were determined by dideoxy chain termination method. DNASTAR software was used to analyze the homology of 13 ’end sequence of LSA from different isolates. [Result] The specific FCC1 / HNLSA 13 ’end sequence was obtained by PCR amplification. The correct pT LSA 1 recombinant plasmid was obtained by restriction enzyme digestion and PCR. Sequencing showed that the 13 ’end of the cloned LSA was 795bp in size and encoded 246 amino acids. Sequence analysis showed that there were only 3 different sites in the amino acid sequence of the FCC1 / HN strain of P. falciparum and the 13 ’end of LSA of NF5 4, T9 / 96, KEN, PNG and BRA strains in China. 【Conclusion】 The 13 ’end fragment of Plasmodium falciparum FCC1 / HNLSA was cloned. Sequence analysis and homology analysis showed that the 13 ’end sequence of FCC1 / HNLSA has high homology with other isolates.