应用四环素调控系统建立可调控抗恶性疟原虫DNA疫苗的初步研究

Preliminary study on regulable DNA vaccines against Plasmodium falciparum

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 。

AIM: To construct regulable DNA vaccine against Plasmodium falciparum by using tetracycline(Tet) regulable system. METHODS: Eukaryotic expression vectors pTL 8/apical membrane antigen 1 (AMA 1) (tTA) and pTL 8/AMA 1(rtTA)gene which express trans activator (tTA) or reverse trans activator(rtTA), respectively, and AMA 1 gene of Plasmodium falciparum were constructed. BALB/c mice were immunized with these plasmids and doxycycline (dox) was administered to regulate the expression of AMA 1. For some mice immunized with pTL 8/AMA 1(rtTA), pUHS6 1, a plasmid containing trans silencer (tTS) to suppress basal expression of AMA 1 from pTL 8/AMA 1(rtTA), was injected into these mice together with pTL 8/AMA 1(rtTA). The sera of the mice were isolated at 2,4,6 and 8 weeks post immunization and the antibodies specific to AMA 1 were measured by ELISA. RESULTS: pTL 8/AMA 1 and pTL 8/AMA 1(rtTA) were constructed successfully. The mice immunized by pTL 8/AMA 1(tTA) with dox or by pTL 8/AMA 1(rtTA) without dox (at these conditions, AMA 1 was expressed at basal level)developed significant antibodies against AMA 1. Mice immunized by pTL 8/AMA 1(rtTA)and pUHS6 1 without dox did not develop significantly antibodies against AMA 1. In contrast, the mice immunized by pTL 8/AMA 1(rtTA)and pUHS6 1 with dox produced high level of antibodies. CONCLUSION: pTL 8/AMA 1(rtTA)combined with pUHS6 1 is a good regulable DNA vaccine candidate against Plasmodium falciparum .