双启动子DNA疫苗载体pCMVnir的构建及其启动子的活性

目的：研制携带CMVie和NirB两种双启动子的DNA疫苗新型载体pCMVnir(pCN)。方法：设计细菌IVI启动子nirB，置换pTfiEx-4中plO和T71ac启动子，获得pCN，将报告基因pEGFP-N1和pDsRed2-N1分别插入pCN构建成pCN—EGFP和pCN—DsRed两种报告质粒，转化减毒沙门菌Salmonella SL3261和大肠杆菌DH5d；同时采Fugene脂质体，转染Hela细胞和前列腺细胞，检测pCN启动子的活性。结果：pCN的两个启动子都可高效转录和表达两种荧光报告基因，并且nirB是温和厌氧依赖性的。结论：成功构建了一种双启动子DNA疫苗表达载体pCN。

Construction and evaluation of new dual-promoter DNA vaccine expression plasmid pCMVnir with in vivo-inducible bacterial nirB and CMVie promoters

Objective: To obtain a new dual-promoter DNA vaccine expression plasmid pCMVnir with in vivo-inducible bacterial CMVie and nirB promoters. Methods: Native nirB promoter was derived from E.coli, and a modified version of nirB promoter was designed and synthesized by deleting the nir-response element, whose activation depends on low-level of oxygen tension. In order to study the activity of these promoters, two different reporter genes EGFP-N1 and Ds-Red were inserted into the pCMVnir. Both pCN-EGFP and pCN-DsRed reporter plasmids were transfected into attenuated Salmonella and E.coli DH5to naked eyes. When the bacteria were cultured on the LB agar containing ampicillin, the colonies became lightly red and green. The expression strength of the reporter genes were confirmed by the flow cytometer, and the data indicated that nirB was activated at normal condition. Conclusion: The pCMVnir promoter could express antigens in vivo, and it is suitable for developing intracellular bacteria-based DNA vaccine, so it can express the antigen genes in both intracellular bacteria and mammalian cells, and has the capacity to enhance the immunogenicity of DNA vaccine.