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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pTriEx4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pCNEGFP和pCNDsRed两种报告质粒,分别转化S.SL3261、E.coliDH5α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。pCNEGFP转化S.SL3261,接种BALBc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ELISA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pCN16L1E7及对照单启动子载体pCMV16L1E7和pNir16L1E7,对比三者诱导黏膜免疫的差别。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pCMVnir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pCNEGFP诱导的免疫反应高于常用的单启动子载体pCMVEGFP。人乳头瘤病毒双启动子载体pCN16L1E7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。
Objective To construct a novel double promoter expression vector pCMVnir and study the effect of its combination with intracellular parasites on the promotion of gene immunity. Methods The bacterial nitrate reductase gene B promoter nirB was an anaerobic promoter. According to the structure of nirB promoter, an improved nirB was designed and synthesized, and the T7Lac and P10 promoters in the three promoter pTriEx4 were replaced, while their CMVie promoter A new vector, pCMVnir, which carries both CMVie and nirB promoters, or pCN was obtained. Two reporter plasmids, pCNEGFP and pCNDsRed, were constructed and transformed into the cells of S.SL3261, E.coli DH5α and CHO respectively to detect the expression of the reporter gene to determine the activity of the two promoters. pCNEGFP was transformed into S.SL3261, BALBc mice were inoculated, the mice were dissected regularly to collect the mucosa-associated lymphoid tissues, and the colonization and stability of recombinant bacteria and vector DNA in the cells were observed. Mice were immunized, and mice sera and vaginal secretions were collected periodically. Recombinant rEGFP was used as an antigen and its ability to enhance the immune response was investigated by ELISA. The human papillomavirus L1E7 double promoter pCN16L1E7 and the control single promoter vector pCMV16L1E7 and pNir16L1E7 were constructed to compare the differences of mucosal immunity induced by the three. Results The double promoter expression vector pCMVnir effectively expressed the reporter gene in bacteria and eukaryotic cells. Fluorescent colonies and precipitates were observed in the bacteria. Eukaryotic cells transfected with CHO were able to express the fluorescent protein, indicating that the pCMVnir vector All kinds of promoters are active, of which nirB activity is regulated by facultative anaerobic. pCMVnir has good compatibility with bacteria, and the recombinant bacteria can be replicated and colonized for a limited period of 6 weeks in animals. The plasmid can stably exist in bacteria. The detection of rEGFP antigen showed that pCNEGFP-induced immune response was higher than that of the common single promoter vector pCMVEGFP. Human papillomavirus double promoter vector pCN16L1E7 also induced higher immune responses than other single promoter vectors. Conclusion The double promoter expression vector pCMVnir can be used to match with the attenuated intracellular parasites, which can increase the antigen expression level and enhance the intensity of the gene immune response. And can be used as a general gene cloning and expression vector, especially as a prokaryotic expression vector, without adding inducer, you can get a lot of expression.