恶性疟原虫子孢子表面蛋白2基因的克隆与序列分析

目的：克隆恶性疟原虫海南株(FCC1／HN)子孢子表面蛋白2(PfSSP2)基因，并进行序列分析。方法：根据PfSSP2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中扩增出PfSSP2基因，并克隆入pMD-18T测序载体。用双脱氧链末端终止法测序，用DNAstar软件辅助分析基因结构及进行同源性比较。结果：PCR扩增得到特异的FCC1／HN株PfSSP2基因序列，酶切及PCR鉴定获得了正确的pT-PfSSP2重组质粒。测序表明，所克隆的PfSSP2基因大小为1680bp，编码559个氨基酸残基。序列分析表明，我国恶性疟原虫FCC1／HN株与国外的3D7、DD2、FCR3、HB3A、K1、Thai814、Thai838、Thai842、Thai947及7G8株PfSSP2的氨基酸序列在33个位点存在氨基酸替代，1处氨基酸序列增加；各株间PfSSP2的氨基酸序列同源性都在95.7％以上。结论：克隆了恶性疟原虫FCC1／HN株PfSSP2基因。序列测定及同源性分析表明，恶性疟原虫不同分离株间有高度的同源性。

Cloning and Sequence Analysis of PfSSP2 Gene of Plasmodium falciparum

Objective To clone and determine the nucleotide sequence of the sporozoite surface protein 2(PfSSP2) gene of Plasmodium falciparum isolate FCC1/HN, and to compare the sequences of PfSSP2 genes of different isolates. Methods The PfSSP2 gene was amplified by PCR from genomic DNA of P.falciparum isolateFCC1/HN. Then it was cloned into pMD18 T vector by T A cloning method. The recombinant plasmid was used as template, and the nucleotide sequence of PfSSP2 gene was determined by the dideoxy chain termination method. The DNAstar software was used to analyze the homology of PfSSP2 genes among different P.falciparum isolates. Results The PfSSP2 gene of P.falciparum isolate FCC1/HN was specifically amplified, and the correct recombinant plasmid pT PfSSP2 was constructed. The result of nucleotide sequencing showed that the PfSSP2 gene of P.falciparum isolate FCC1/HN was 1680 base pairs in length, encoding 559 amino acids. The PfSSP2 antigens of P.falciparum isolates FCC1/HN, 3D7, DD2, FCR3, HB3A, K1, Thai814, Thai838, Thai842, Thai947 and 7G8 exhibited 33 amino acid substitutions and 1 region of amino acid additions, with over 95.7% homology in the overall amino acid sequences. Conclusion The PfSSP2 gene of P.falciparum isolates FCC1/HN was successfully cloned and sequenced. The PfSSP2 antigen FCC1/HN and other isolates shared quite high homology.