恶性疟原虫红内期p41-3基因真核表达重组质粒的构建及序列分析(英文)  

Construction of Eukaryotic Expression Recombinant Plasmid and Sequence Analysis of p41-3 Gene of Plasmodium Falciparum Isolate FCC1/HN

作　　者：单志新[1] 余新炳[1] 李学荣[1] 马长玲[1] 陆家海[1] 徐劲[1] 

机构地区：[1]中山医科大学寄生虫学教研室,广东广州510089

出　　处：《中山医科大学学报》2001年第5期329-334,共6页Academic Journal of Sun Yat-sen University of Medical Sciences

基　　金：EducationCommissionofChinafordoctoral trainingunits(93 186) ;Guangdongprovincialnaturalsciencefoundation(980 0 89) ;SunYat senUniversityofMedicalSciences foundation(98169)

摘　　要：【目的】构建恶性疟原虫海南 (FCC1/HN)株 p41 3基因真核表达重组质粒pcDNA3 p41 3;测定p41 3基因序列 ,并了解FCC1/HN株与其它分离株 p41 3序列的差异。【方法】根据 p41 3基因已知序列设计合成 2对引物 ,用PCR技术从FCC1/HN株基因组DNA中扩增 p41 3基因 ;将p41 3基因定向克隆入真核表达载体 pcDNA3 ,转化大肠杆菌DH5α感受态细胞 ;用酶切 ,PCR扩增鉴定筛选到的重组质粒阳性克隆。以正确的重组质粒为模板 ,用双脱氧链末端终止法测定 p41 3基因序列 ,应用软件辅助分析p41 3序列及进行同源性比较。【结果】PCR扩增得到特异的FCC1/HN株 p41 3基因 ;正确的 pcD NA3 p41 3重组质粒被筛选和鉴定。测序表明 ,FCC1/HN株p41 3基因大小为 2 137bp ,编码 375个氨基酸。恶性疟原虫FCC1/HN株与FCBR株 p41 3基因核苷酸序列同性为 98.98% ,编码氨基酸序列同源性为 99.73%。【结论】从恶性疟原虫FCC1/HN株基因组DNA中获取p41 3基因 ,成功构建真核表达重组质粒pcDNA3 p41 3,并测定了FCC1/HN株p41 3基因的序列 ;FCC1/HN株与其它分离株 p41To construct a eukaryotic expression plasmid containing a gene encoding a 41 3 kilodalton blood stage antigen (p41 3) of Plasmodiu falciparum isolate FCC1/HN, and to determine the sequence of p41 3 gene and analyze the homology of the sequences of p41 3 gene of different P.falciparum isolates. Two pairs of primers were designed according to the known sequence of p41 3 gene.Using PCR technique, the p41 3 gene was obtained by amplification from genomic DNA of isolate FCC1/HN. By cloning target gene into a eukaryotic expression vector, pcDNA 3, a recombinant plasmid pcDNA 3 p41 3 was constructed and transferred into E.coli DH5α. The positive clones were screened and identified by agarose gel electrophoresis, endonuclease digestion and PCR technique. The correct recombinant plasmid pcDNA 3 p41 3 was used as template,and the nucleotide sequence of p41 3 gene was determined by the dideoxy chain termination method. Using softwares to analyze the structure and sequence homology of p41 3 gene between isolate FCC1/HN and FCBR. The p41 3 gene was specifically amplified from genomic DNA of Plasmodiumm falciparum isolate FCC1/HN,and the correct recombinant plasmid pcDNA 3 p41 3 was screened and identified. The result of sequence determination showed that the p41 3 gene of isolate FCC1/HN was 2 137 base pairs in full length,encoding 375 amino acids.Isolate FCC1/HN and isolate FCBR exhibited 98 98% homology in the nucleiotide sequences and 99 73% homology in the encoded amino acids of p41 3 gene. [Conclusion] The eukaryotic expression plasmid pcDNA 3 p41 3 is successfully constructed and nucleotide sequence of p41 3 gene of isolate FCC1/HN is determined. The p41 3 genes of isolate FCC1/HN and isolate FCBR share quite high homology.

关 键 词：恶性疟原虫 红内期p41-3基因 重组质粒 构建 序列分析 真核表达 

分 类 号：R382.31[医药卫生—医学寄生虫学]