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目的 构建恶性疟原虫海南分离株 ( FCC1/ HN)裂殖子表面蛋白 2 ( MSP2 )融合 HBs Ag基因片断真核表达质粒 p VX-ORF1- Pf MSP2 - HBS及重组真核表达质粒 p VXORF1- Pf MSP2 ,为疟疾核酸疫苗及蛋白疫苗的研制奠定基础。 方法 ( 1)采用 PCR技术对恶性疟原虫 FCC1/ HN株 MSP2 原核表达质粒 PET2 8α- MSP2 的 MSP2 基因进行扩增 ,扩增产物经纯化后用 Bam H 酶切 ;质粒 p VXORF1- Pv MSP1 .1 9- HBS用相同的酶酶切 ,经纯化后用 T4DNA连接酶将其与酶切后纯化的 MSP2 基因连接 ;( 2 )分别用Bam H +Xho 双酶切 PET2 8α- MSP2 质粒 DNA和 p VXORF1质粒 DNA,纯化后将目的基因片断用 T4DNA连接酶连接 ;将 ( 1)、( 2 )连接产物分别转化大肠杆菌 DH5α。于氨苄青霉素阳性 L B培养平板上筛选阳性克隆 ,酶切电泳鉴定。 结果 筛选出的重组子为编码 FCC1/ HN MSP2 基因片断的重组质粒 p VXORF1- Pf MSP2 - HBS及 p VXORF1- Pf MSP2 。 结论 编码 FCC1/ HN MSP2 基因片断真核表达质粒 p VXORF1- Pf MSP2 - HBS和 p VXORF1- Pf MSP2 的构建为疟疾核酸疫苗及蛋白疫苗的研制奠定了基础。
Objective To construct the eukaryotic expression plasmid pVX-ORF1-Pf MSP2 - HBS and the recombinant eukaryotic expression plasmid p VXORF1-Pf (MSP2) fused to HBsAg gene of Plasmodium falciparum Hainan isolate (FCC1 / HN) MSP2, laid the foundation for the development of malaria nucleic acid vaccine and protein vaccine. Methods (1) The MSP2 gene of Plasmodium falciparum FCC1 / HN strain MSP2 prokaryotic expression plasmid PET2a-MSP2 was amplified by PCR. The amplified product was purified and then digested with Bam H. Plasmid p VXORF1-Pv MSP1.1 9-HBS was digested with the same enzymes, purified and ligated with the purified MSP2 gene after digestion with T4 DNA ligase; (2) Double-digested PET2a-MSP2 plasmid DNA and pVXORF1 The plasmid DNA was purified, and the target gene fragment was ligated with T4 DNA ligase. The ligated products of (1) and (2) were respectively transformed into E. coli DH5α. Positive clones were screened on ampicillin-positive LB medium and identified by restriction enzyme digestion. Results The recombinant plasmids pVXORF1 - Pf MSP2 - HBS and p VXORF1 - Pf MSP2 were selected as the recombinants screened for the FCC1 / HN MSP2 gene. Conclusion The construction of eukaryotic expression plasmids pVXORF1 - Pf MSP2 - HBS and p VXORF1 - Pf MSP2 encoding the fragment of FCC1 / HN MSP2 laid the foundation for the development of malaria nucleic acid vaccine and protein vaccine.