Aim To construct a prokaryotic expression plasmid contained agene encoding membrane-associated calcium-binding protein (Pfs40) of Plasmodium falciparum isolate FCC1/HN,sequence the gene of Pfs40 and find out the diffences of the Pfs40 gene sequence between this isolate and other isolate. Methods One pair of primers were designed according to the known sequence of PFS40 gene.with PCR technique an encoding region of Pfs40 gene was obtained by amplification of genomic DNA of isolate FCC1/HN.The amplified product was identified by EcoRⅤ.By cloning target gene into a prokaryotic expression vector,PET28a,a recombinant PET28a-Pfs40 was constructed and transfomed into E.coli DH5α.The nucleotide sequence of the Pfs40 gene was determined by the dideoxy chain termination method.The using of softwares to analyzd the Pfs40 gene sequence and deducing amino acid sequence. Results The Pfs40 gene with about 1032 base pairs was specifically amplified in accordance with expected one.The positive recombinant PET28a-Pfs40 was screened and identified by agarose gel electrophoresis ,endonuclease digestion and PCR technique.Isolate FCC1/HN and isolate 7G8 exhibited 99.5% homology in nucleotide sequences and 99.1%homology in the amino acid sequences.Five calcium-binding EF-hand loops and four marked antigenic sequential epitopes were determined in the deduced amino acid sequences of Pfs40 gene of isolate FCC1/HN. Conclusion The gene encoding Pfs40 was amplified from genomic DNA of Plasmodiumm falciparum and PET28a-Pfs40 recombniant was successfully constructed.The Pfs40 genes of isolate FCC1/HN and isolate 7G8 shared high homology.%目的 构建恶性疟原虫海南(FCC1/HN)株膜相关钙结合蛋白(Pfs40)基因编码区的原核表达载体,测定Pfs40基因编码区的序列,了解该虫株与其它虫株Pfs40基因序列的差异。方法 根据Pfs40基因已知序列设计合成一对引物,用PCR技术从FCC1/HN株基因组DNA中扩增Pfs40基因编码区;EcoRⅤ酶切鉴定PCR产物；将Pfs40基因插入原核表达载体PET28a，转化大肠杆菌DH5α感受态细胞，于卡那阳性LB培养平板上筛选阳性克隆，酶切，PCR扩增鉴定；用双脱氧链末端终止法进行测序，应用软件对Pfs40核苷酸及推测氨基酸序列进行分析。结果 PCR扩增获得1032bp的Pfs40基因，EcoRⅤ酶切表明扩增产物正确；重组质粒经双酶切及PCR鉴定表明获得正确重组子；恶性疟原虫FCC1/HN株与7G8株Pfs40基因核苷酸序列同源性为99.5%，编码氨基酸序列同源性为99.1%。Pfs40理论蛋白质有5个钙结合区EF-hand结构；4个明显的抗原表位区段。结论 从恶性疟原虫基因组DNA中获取Pfs40基因，并成功构建恶性疟原虫FCC1/HN株Pfs40基因编码区的原核表达载体；FCC1/HN株与7G8株Pfs40基因有高度的同源性。
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