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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。
Objective: To construct the Pseudomonas aeruginosa FCC1 / HN isolates of P. falciparum, which has the sex - specific antigen Pfs48 / 45 and provide the antigen for the development of the Plasmodium falciparum transmission block vaccine. Methods: Primers were designed according to the coding region of Pfs48 / 45 gene. The DNA was amplified by PCR and sequenced. After double enzyme digestion, it was cloned into pcDNA3 vector and transformed into E. coli TG1. Ampicillin resistant colonies were randomly removed for PCR amplification Increased by alkali lysis positive extraction of recombinant DNA, double enzyme digestion. Results: The full-length Pfs48 / 45 gene (1363bp) was amplified by PCR and 450 bases were detected by 5-terminal oligonucleotide primer. The results showed that the sequence of Pfs48 / 455 FCC in FCC1 / HN isolate was similar to NF54 The corresponding genes were basically the same, only the bases 307 (T → C) and 372 (T → C) were replaced; the 372th base was substituted to generate a new restriction enzyme TaqI, and the PCR product was further digested with TaqI , Resulting in two 984bp and 379bp digested fragments respectively. Sequencing and digestion results confirmed that the target gene was indeed Pfs48 / 45 gene. The purified target gene fragment was inserted into the BamHI and EcoRI sites of pcDNA3 plasmid point. CONCLUSION: The sequence of Pfs48 / 45 antigen of FCC1 / HN isolate in Hainan is highly homologous to the corresponding gene of NF54 strain. The recombinant plasmid pcDNA3-Pfs48 / 45 was successfully constructed.