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目的 :应用四环素 (tetracycline,Tet)可调控系统建立可调控的抗恶性疟原虫的DNA疫苗。方法 :首先构建恶性疟原虫顶端膜抗原 1(AMA 1)基因和转录激活因子 (tTA或rtTA)基因的真核表达质粒pTL 8/AMA 1和pTL 8/AMA 1(rtTA) ,并大量制备这两种质粒及表达转录抑制子 (tTS)的质粒pUHS6 1。以pTL 8/AMA 1、pTL 8/AMA 1(rtTA)和pTL 8/AMA 1(rtTA)加pUHS6 1/tTS免疫小鼠后 ,用四环素类似物强力霉素 (doxcycline ,dox)诱导或抑制上述DNA载体内AMA 1的表达 ,并分离小鼠血清检测针对AMA 1抗体的表达情况。结果 :①构建了真核表达质粒pTL 8/AMA 1和pTL 8/AMA 1(rtTA) ;②pTL 8/AMA 1和pTL 8/AMA 1(rtTA)在没有被诱导表达时 (仅基础表达 ) ,可诱导明显的免疫应答。在以pTL 8/AMA 1(rtTA)与含tTS的pUHS6 1共同免疫后 ,可极大地降低其免疫应答。应用dox诱导pTL 8/AMA 1(rtTA)中的AMA 1基因表达后 ,可明显引起免疫应答。结论 :pTL 8/AMA 1(rtTA)附加pUHS6 1构成了可调节的DNA免疫系统。该系统的建立将对进一步深入研究DNA疫苗的免疫机制和调控基因表达 ,减少不良免疫反应以及进行可精确调控的基因治疗打下一定的基础
OBJECTIVE: To establish a DNA vaccine against Plasmodium falciparum that can be regulated by tetracycline (Tet) regulatory system. Methods: The eukaryotic expression plasmids pTL 8 / AMA 1 and pTL 8 / AMA 1 (rtTA) of Plasmodium falciparum apical membrane antigen 1 (AMA 1) gene and transcription activator (tTA or rtTA) gene were constructed and prepared in large quantities Two plasmids and plasmid pUHS6 expressing transcriptional repressor (tTS) 1. Following immunization of mice with pTL8 / AMA1, pTL8 / AMA1 (rtTA) and pTL8 / AMA1 (rtTA) plus pUHS6i / tTS, the above described DNA vector expression of AMA 1, and mouse serum was isolated for the detection of AMA 1 antibody expression. Results: ① The eukaryotic expression plasmids pTL 8 / AMA 1 and pTL 8 / AMA 1 (rtTA) were constructed. ② The expression of pTL 8 / AMA 1 and pTL 8 / AMA 1 (rtTA) Can induce a significant immune response. After co-immunization with pTL8 / AMA1 (rtTA) with pTSH1 containing tTS, its immune response can be greatly reduced. Dox-induced AMA1 gene expression in pTL8 / AMA1 (rtTA) evoked a significant immune response. Conclusion: pTL 8 / AMA 1 (rtTA) plus pUHS6 1 constitutes an adjustable DNA immune system. The establishment of this system will lay a certain foundation for further research on the immune mechanism of DNA vaccine and regulation of gene expression, reduction of adverse immune response and gene therapy with precise regulation