恶性疟原虫有性期特异抗原Pfs48/45基因的克隆与部分序列分析

中国寄生虫学与寄生虫病杂志 ›› 1998, Vol. 16 ›› Issue (6): 406-410.

恶性疟原虫有性期特异抗原Pfs48/45基因的克隆与部分序列分析

罗树红; 余新炳; 刘彦文; 方建民; 徐劲; 李学荣

中山医科大学寄生虫学教研室

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:1998-12-31 发布日期:1998-12-31

CLONING AND SEQUENCING OF THE GENE CODING THE SEXUAL STAGE ANTIGEN Pfs48/45 OF PLASMODIUM FALCIPARUM

Luo Shuhong; Yu Xinbing; Liu Yanwen; Fang Jianming; Xu Jing; Li Xuerong

Department of Parasitology; Sun Yat sen University of MedicalSciences; Guangzhou 510089

Received:1900-01-01 Revised:1900-01-01 Online:1998-12-31 Published:1998-12-31

摘要/Abstract

摘要： 目的：构建恶性疟原虫海南FCC1／HN分离株有性期特异抗原Pfs48／45，为研制恶性疟原虫传播阻断疫苗提供抗原。方法：根据Pfs48／45基因编码区序列设计引物，用PCR扩增DNA，并进行序列分析，双酶切后，定向克隆入pcDNA3载体，转化大肠杆菌TG1，随机取出氨苄青霉素抗性菌落进行PCR扩增，用碱裂解法抽提阳性的重组子DNA，双酶切鉴定。结果：特异扩增了编码Pfs48／45全基因序列（1363bp）；用5端寡核苷酸引物测定了450个碱基，结果表明FCC1／HN分离株Pfs48／455端基因序列与NF54株相应基因基本一致，仅在第307（T→C）和372（T→C）位碱基存在置换；第372位碱基置换产生新的酶切位点TaqI，进一步用TaqI消化PCR扩增产物，产生两个大小分别为984bp和379bp的酶切片段，测序和酶切结果均证实扩增的目的基因确为fs48/45 基因; 将纯化的目的基因片段正向插入pcDNA 3 质粒的BamHI 和EcoRI 位点。结论: 我国海南FCC1/ HN 分离株Pfs48/45 抗原基因序列与NF54 株相应基因高度同源; 成功构建重组质粒pcDNA 3-Pfs48/45

关键词: 恶性疟原虫, 传播阻断免疫, 克隆, DNA序列分析, 疟疾疫苗

Abstract: AIM: To express the antigen Pfs48/45 in vitro and provide an antigen for the
development of the transmission blocking vaccine. METHODS:According to the published nucleotide
sequence of Pfs48/45 of Plasmodium falciparum isolate NF54, a pair of oligonucleotides was
designed and used as primers(P1,P2). The gene encoding the gametocyte/gamete specific membrane
protein Pfs48/45 of P. falciparum isolate FCC1/HN has been amplified by using polymerase chain
reaction(PCR) technique. The PCR product was purified and directly sequenced by the dideoxynucleotide terminator method with 5’-end primer P1. At the same time, the purified PCR product was digested with BamHI and EcoRI and cloned into the plasmid pcDNA 3, then the recombinant clones were transformed into E. coli strain TG1. The recombinant plasmid pcDNA 32Pfs48/45 was screened and identified by PCR amplification and restriction analysis. RESULTS: ① The gene fragment Pfs48/45 was specifically amplified from the genomicDNA of Plasmodium falciparum isolate FCC1/HN; ②The sequence demon-strated that the 5’-end nucleotide and predicted aminoacid sequence of Pfs48/45 from FCC1/HN isolate was basically identical with that from NF54 isolate. We found that the sequence of the Pfs48/45 gene from the isolate FCC1/HN differs from the published sequence (isolate NF54) only at positions 307 and 372 (T →C). The substitution of T →C at the position of 372 generates a new restriction site Taq I. The PCR product digested by Taq I generates DNA fragment of 984 bp and 379 bp , suggesting that the PCR product is the gene encoding the transmission blocking antigen Pfs48/45; ③ The gene fragment of
Pfs48/45 was directly inserted into the BamHI and EcoRI site of plasmid pcDNA 3. CONCLUSION: The nucleotide sequence of Pfs48/4 5 of Plasmodium falciparum isolate FCC1/HN from south China was similar to that of isolate NF54. The recombinant plasmid pcDNA 3 Pfs48/45 was successfully constructed, providing a means to evaluate the role and biological function of this sexual-stage-specific protein of Pfs48/45.

Key words: Plasmodium falciparum, transmission blocking immunity, clone DNA sequence analysis, malaria vaccine

罗树红;余新炳;刘彦文;方建民;徐劲;李学荣. 恶性疟原虫有性期特异抗原Pfs48/45基因的克隆与部分序列分析[J]. 中国寄生虫学与寄生虫病杂志, 1998, 16(6): 406-410.

LuoShuhong;YuXinbing;LiuYanwen;FangJianming;XuJing;LiXuerong. CLONING AND SEQUENCING OF THE GENE CODING THE SEXUAL STAGE ANTIGEN Pfs48/45 OF PLASMODIUM FALCIPARUM[J]. Chinese Journal of Parasitology and Parasitic Diseases, 1998, 16(6): 406-410.

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