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

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

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Ｐｆｓ４８／４５基因；将纯化的目的基因片段正向插入ｐｃＤＮＡ３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ｐｃＤＮＡ３－Ｐｆｓ４８／４５。

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 pcDNA3, then the recombinant clones were transformed into E.coli strain TG1. The recombinant plasmid pcDNA3 Pfs48/45 was screened and identified by PCR amplification and restriction analysis. RESULTS:① The gene fragment Pfs48/45 was specifically amplified from the genomic DNA of Plasmodium falciparum isolate FCC1/HN;②The ssequence demon strated that the 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 pcDNA3.CONCLUSION:The nucleotide sequence of Pfs48/45 of Plasmodium falciparum isolate FCC1/HN from south China was similar to that of isolate NF54. The recombinant plasmid pcDNA3 Pfs48/45 was successfully constructed, providing a means to evaluate the role and biological function of this sexual stage specific protein of Pfs48/45.