恶性疟原虫FCC1/HN株RESA基因克隆及序列分析

[目的 ]测定恶性疟原虫海南株 (FCC1/HN)环状体感染红细胞表面抗原 (RESA)基因 3′端部分基因序列 ,比较FCC1/HN与国外分离株RESA序列的差异。 [方法 ]应用PCR技术扩增RESA基因 3′端部分序列 ,将其克隆入pMD18 T载体。阳性重组克隆经酶切及PCR鉴定后 ,用双脱氧链末端终止法进行基因序列测定 ,并用分子生物学软件进行基因结构和同源性分析。 [结果 ]用PCR成功扩增出约 846bp的RESA基因特定片段 ,阳性克隆经酶切及PCR扩增确定。基因序列分析表明 ,我国恶性疟原虫FCC1/HN株与国外FC2 7,PaloAlto ,NF7株RESA基因序列有不同程度的差异。 [结论 ]确定了恶性疟原虫FCC1/HN株RESA基因 3′端序列。同源性分析表明 ,FCC1/HN株RESA序列与其他分离株存在一定差异

CLONING AND SEQUENCE ANALYSIS OF RESA GENE FRAGMENT OF PLASMODIUM FALCIPARUM ISOLATE FCC1/HN

OBJECTIVE: To determine the nucleotide sequence of the 3'-termal of the RESA gene Plasmodium falciparum isolate FCC1/HN, and find out the differences of the sequences of RESA gene among isolate FCC1/HN, FC27, NF7 and Palo Alto. METHODS: 3'-terminal fragment of RESA gene of P. falciparum isolate FCC1/HN was amplified by PCR method, then was cloned into pMD18-T vector. The recombinant was screened and identified by BamHI + XhoI and PCR technique. The nucleotide sequence of the 3'-terminal of the RESA gene was determined by the dideoxy chain termination method. DNASTAR and BLAST software were used to compare and analyze the RESA gene sequences among the different isolates. RESULTS: The 3'-termal fragment of the RESA gene with about 846 bp was specifically amplified by PCR, the recombinant pMD18-T-RESA was successfully constructed. Different degrees of diversity of the RESA gene sequences were found among P. falciparum isolates FCC1/HN, FC27, NF7 and Palo Alto. CONCLUSION: There were differences in the sequences of RESA gene among the P. falciparum isolate FCC1/HN and three other isolates (FC27, NF7 and Palto alto).