新型双启动子表达质粒pCMVnir的构建及其促进基因免疫效果的研究

目的 构建一种新型双启动子表达载体pCMVnir，研究其与胞内寄生菌联合应用促进基因免疫效果的作用。方法细菌硝酸盐还原酶基因B启动子nirB为厌氧诱导启动子，根据nirB启动子的结构，设计合成改良的nirB，置换三启动子载体pTriEx-4中的T7Lac和P10启动子，而保留其CMVie启动子，获得携带CMVie和nirB双种启动子的新型载体pCMVnir，或称为pCN。构建pCN-EGFP和pCN-DsRed两种报告质粒，分别转化S．SL3261、E．coil DH-15a和CHO等细胞，检测报告基因的表达情况以确定两种启动子的活性。pCN-EGFP转化S．SL3261，接种BALB／c小鼠，定期解剖小鼠收集黏膜相关淋巴组织，观察重组细菌和载体DNA在细胞中的定植及稳定性。免疫接种小鼠，并定期收集小鼠血清和阴道分泌物，以重组rEGFP为抗原，用EEISA方法研究其增强免疫应答的能力。并构建人乳头瘤病毒L1E7双启动子pCN-16L1E7及对照单启动子载体pCMV-16L1E7和pNir-16L1E7，对比三者涛导黏膜免疫的差别。结果双启动子表达载体pCMVnir可在细菌和真核细胞中有效表达报告基因，在细菌中可形成肉眼可见的荧光菌落和沉淀，转染CHO等真核细胞能够表达荧光蛋白，表明pC-MVnir载体的两种启动子都有活性，其中nirB活性是受兼性厌氧调控的。pCMVnir与细菌有较好的相容性，重组细菌可在动物体内有限复制和定植达6周，质粒可在细菌中稳定存在。以rEGFP为抗原检测发现pCN-EGFP诱导的免疫反应高于常用的单启动子载体pCMV-EGFP。人乳头瘤病毒双启动子载体pCN-16L1E7诱导的免疫反应也高于其他单启动子载体。结论双启动子表达载体pcMVnir与减毒胞内寄生菌匹配应用，能够提高抗原表达量及增强基因免疫反应的强度。并可作为一般基因克隆和表达载体，特别是作为原核表达载体，不用添加诱导物，即可获得大量表达。

Construction of a dual-promoter plasmid pCMVnir with HCMVie and in vivo-inducible bacterial nirB promoters and its advantages in genetic immunization

Objective To develop a plasmid matching the bacteria-based DNA vaccine inoculation. Methods Dual-promoter DNA vaccine with eukaryotic HCMVie and prokaryotic promoter nirB were designed to improve antigen expression in vivo by replacing the T71ac and P10 promoters of pTriEx-4 and keeping the CMVie promoter of pTriEx-4. The nirB promoter was derived from E. coli, a modified version of nirB promoter without nitrogen-response element; its activation depends only on low-level of oxygen tension. We constructed dual-promoter expression plasmid pCN-16L1E7 and the other two single-promoter vectors pCMV-16L1E7 and pNir-16L1E7, and detected the mucosal immune reactions induced by these vector-transformed Salmonella strains. Results The pCMVnir plasmid is successfully developed. In addition, the recombinant Salmonella could rephcate in vivo up to 6 weeks, especially in mucosa-associated lymph organs and antigen-processing cells. The nirB promoter could be activated by aerobic and anaerobic condition, high expression of antigen did not interfere the stability of the recombinant bacteria. Immune reaction induced by pCN-EGFP is higher than that of pCMV-EGFP. Dual-promoter pCN-16L1E7 could induce stronger immune reaction than the other ones. Conclusion pCMVnir is a good candidate vector for developing intracellular bacteria-based DNA vaccines which is more potential for APC Ag presentation and is more immunogenic.