摘要
目的 构建包含恶性疟原虫Pf332基因片段的分泌型、非分泌型真核表达重组质粒。方法 从FCC1/HN株基因组DNA中PCR扩增Pf332基因片段 ,P332 -RO、P332 -R1和P332 -R2 ;用PCR扩增法合成鼠IgG轻链隹号肽的编码序列。用定向克隆法分别构建分泌型重组质粒pcDNA3 -s -P332 -R0、pcDNA3 -s -P332 -R1、pcDNA3 -s -P332 -R2和非分泌型重组质粒pcDNA3 -s -P332 -R0、pcDNA3 -s -P332 -R1、pcDNA3 -s -P332 -R2。阳性克隆的重组质粒DNA经酶切、PCR扩增及测序鉴定。结果 PCR扩增得到特异的FCC1/HN株Pf332基因片段 ,P332 -R0、P332 -R1、P332 -R1,大小分别为 489、42 9和 393bp。用PCR扩增法合成 6 3bp的Balb/c小鼠IgG轻链信号肽的编码序列。酶切、PCR及测序鉴定表明获得了正确的含Pf332基因片段的分泌型、非分泌型重组质粒。结论 成功构建分别包含恶性疟原虫Pf332基因片段—P332 -R0、P332 -R1和P332 -R2的分泌型、非分泌型真核表达重组质粒。
Objective To determine the sequence of exported protein 1(exp-1) gene of Plasmodium falciparum(P. f )isolate FCC 1/HN. Methods According to the known sequence of a pair of primers were designed and synthesized. The exp-1 gene was amplified by polyrnerase chain reaction(PCR) from genomic DNA of isolate FCC I /HN and cloned into the pMD1-18T vector. Then the recombinant plasmid pT-exp-1 was transformed into E. coli JM109. The positive clones were screened and identified by agarose gel electrophoresis endonuclease digestion and PCR technique. The correct recombinant plasmid pT-exp-1 was used as template and the nucleotide sequence of exp-1 gene was determined by the dideoxy chain termination method. Results The exp-gene from genomic DNA of P. falciparum isolate FCC l/HN was specifically amplified, and the correct recombinant plasmid pT-exp-1 was constructed . The full length of exp-1 gene of isolate FCC1/HN, encoding 162 amino acids is 937 base pairs . Conclusion The exp-1 gene of P. falciparum was cloned and sequenced , and a foundation was laid for further study on the function of exp-1 gene.
出处
《中国热带医学》
CAS
2001年第4期294-298,共5页
China Tropical Medicine
基金
中山医科大学"2 1 1"重点学科建设课题基金 (981 69)
广东省自然科学基金 (980 0 89)
教育部博士点基金(93 - 1 86)资助
