恶性疟原虫海南株STARP基因真核表达重组质粒的构建及基因结构分析

CONSTRUCTION OF EUKARYOTIC EXPRESSION PLASMID AND STRUCTURE ANALYSIS OF STARP GENE OF PLASMODIUM FALCIPARUM HAINAN ISOLATE

目的 构建恶性疟原虫海南 (FCC1/HN)株STARP基因真核表达重组质粒pcDNA3-STARP ;分析STARP基因结构 ,并了解FCC1/HN株与其它分离株STARP基因序列的差异。方法 根据STARP基因已知序列设计合成两对引物 ,用PCR技术从FCC1/HN株基因组DNA中扩增两个部分序列重叠的STARP基因片段 ;将两基因片段分别双酶切后定向克隆入真核表达载体 pcDNA3 ,转化大肠杆菌DH5α感受态细胞。酶切 ,PCR扩增鉴定筛选重组质粒阳性克隆 ;用双脱氧链末端终止法测序 ,应用软件辅助分析基因结构及进行同源性比较。结果 分别PCR扩增获得 10 78bp ,10 92bp的两个STARP基因片段 ;经双酶切及PCR鉴定表明获得正确重组质粒 ;恶性疟原虫FCC1/HN株与T9/ 96株STARP基因核苷酸序列同源性为 92 .2 % ;推测编码氨基酸序列同源性为 90 .9% ;在STARP蛋白中部重复区推测有多个明显的抗原表位区段。结论 从FCC1/HN株恶性疟原虫基因组DNA中获取STARP基因 ,并成功构建真核表达重组质粒pcDNA3-STARP ;FCC1/HN株与其它分离株STARP基因有高度的同源性。

Aim To construct a eukaryotic expression plasmid contained a gene encoding sporozoite threoine and aspargine rich protein (STARP) of Plasmodiu falciparum isolate FCC1/HN;sequence the gene of STARP and find out the differences of the STARP gene structures of different isolates.Methods Two pairs of primers were designed according to the known sequence of STARP gene.Using PCR technique,two partial STARP genes overlapped were obtained by amplification from genomic DNA of isolate FCC1/HN. By cloning target gene into a eukaryotic expression vector,pcDNA 3,a recombinant pcDNA 3-STARP was constructed and transferred into E.coli DH5α.The nucleotide sequence of the STARP gene was determined by the dideoxy chain termination method.Using softwares to analyze the STARP gene structure and the gene homology between isolate FCC1/HN and T9/96.Results Two partial STARP genes with about 1078,1092 base pairs were specifically amplified in accordance with expected ones.The positive recombinant pcDNA 3-STARP was screened and identified by agarose gel electrophoresis,endonuclease digestion and PCR technique.There are 92.% homology in nuleo tide sequences and 90.9% homology in amino sequences between isolate FCC1/HN and T9/96.A series of marked antigenic sequential epitopes were determined in the repeated amino acids region of the inferred STARP protein of isolate FCC1/HN. Conclusion The gene encoding STARP was amplified from genomic DNA of Plasmodiumm falciparum isolate FCC1/HN and pcDNA 3-STARP recombinant was successfully constructed.The STARP genes of isolate FCC1/HN and T9/96 shared quite high homology.