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目的:构建编码恶性疟原虫复合多价保护性抗原的基因的重组质粒,为进行基因免疫提供条件。方法:设计一对特异引物P1与P2,采用PCR法扩增获取MSP1中C端19肽基因,纯化后用SalⅠ+XbaⅠ双酶切,把含复合基因PfCMR的重组质粒pWR450-1/PfCMR用EcoRⅠ+SalⅠ双酶切,回收复合基因PfCMR,将MSP119基因与PfCMR基因串联后与经EcoRⅠ+XbaⅠ双酶切的真核表达质粒pcDNA3进行重组,转化大肠杆菌JM109,经电泳初筛、双酶切鉴定及PCR鉴定。结果:PCR扩增获得363bp的MSP119基因,重组克隆经双酶切鉴定及PCR鉴定后获得正确重组克隆子pcDNA3-PfCMR-MSP119(命名为pcDNA3-Pf8),Pf8基因长度为618bp。结论:成功构建编码恶性疟原虫多价保护性抗原的基因的重组质粒pcDNA3-Pf8。
OBJECTIVE: To construct a recombinant plasmid encoding the gene encoding multivalent protective antigen of Plasmodium falciparum, which provides conditions for gene immunization. Methods: A pair of specific primers P1 and P2 were designed and synthesized. The C-terminal 19 peptide of MSP1 was amplified by PCR and purified with SalⅠ + XbaⅠ. The recombinant plasmid pWR450-1 / PfCMR containing PfCMR was digested with EcoRⅠ + The gene PfCMR was cut and recovered. The MSP119 gene and PfCMR gene were ligated with the recombinant plasmid pcDNA3, which was digested with EcoRⅠ + XbaⅠ. The recombinant plasmid was transformed into Escherichia coli JM109 and identified by restriction enzyme digestion and PCR. Results: The 363bp MSP119 gene was obtained by PCR amplification. The recombinant clone pcDNA3-PfCMR-MSP119 (named as pcDNA3-Pf8) was double-digested and identified by PCR. The length of Pf8 gene was 618bp. Conclusion: The recombinant plasmid pcDNA3-Pf8 encoding the gene of multivalent protective antigen of Plasmodium falciparum was successfully constructed.