不同组成型启动子对乳酸菌表达系统外源基因表达影响的比较

Comparison of the Effect of Different Constitutive Promoters on Exogenous Gene Expression in Lactobacillus Expression System

【目的】构建不同组成型启动子的乳酸菌表达载体系统,评价不同启动子驱动外源基因表达的活性,筛选出强表达的启动子,为后续研究乳酸菌作为活菌载体表达外源抗原或功能性蛋白提供理论依据。【方法】以大肠杆菌-乳酸菌穿梭载体pPG为骨架,将表达载体中启动子序列分别替换为组成型强启动子Pldh、PHH和Phce,利用PCR方法扩增报告基因增强型绿色荧光蛋白(EGFP)和氨苄青霉素抗性基因(Ampr),并在下游多克隆位点中分别插入荧光素酶(LUC)、EGFP和Ampr报告基因,构建9种乳酸菌表达质粒,电转入副干酪乳杆菌HLJ-27感受态细胞,筛选获得9株重组乳酸菌。通过实时荧光定量PCR检测报告基因在重组菌中的mRNA转录水平,利用Western blotting和间接ELISA检测蛋白的表达量,并检测表达蛋白的活性,以评估3个启动子的活性强弱。【结果】PCR成功扩增出大小约720 bp的EGFP基因和850 bp的Ampr基因。Western blotting结果显示,在9株重组乳酸菌中,分别成功表达出了58 ku的LUC蛋白、26 ku的EGFP蛋白和28 ku的Ampr蛋白。实时荧光定量PCR和间接ELISA检测结果显示,启动子Pldh启动外源基因转录水平和驱动外源蛋白表达水平均显著高于启动子PHH和Phce(P<0.05;P<0.01)。报告基因检测结果显示,启动子Pldh驱动LUC、EGFP和Ampr外源蛋白的活性显著高于启动子PHH和Phce(P<0.05;P<0.01)。【结论】本研究成功构建了3种启动子报告基因表达系统,确定了3种启动子活性强弱依次为Pldh、PHH和Phce,探究出一种启动子活性筛选的优势筛选系统——Ampr报告系统,为高表达乳酸菌载体系统的建立提供了必要的依据。

【Objective】The aim of this study was to construct the lactic acid bacteria expression vector with different constitutive promoters,evaluate the activity of these promoters driving foreign gene expression,and screen out the enhanced expressed promoter with high stability,so as to provide a theoretical basis for the subsequent study that lactic acid bacteria expresses foreign antigens or functional proteins as viable bacterial carriers.【Method】Using the Escherichia coli-lactic acid bacteria shuttle vector pPG as framework,the promoter sequences in the vector were respectively replaced by Pldh,PHH and Phce,enhanced green fluorescent protein(EGFP)and ampicillin resistance gene(Ampr)were amplified by PCR and the reporter genes luciferase(LUC),EGFP and Ampr were respectively inserted into the downstream polyclonal sites of the vector.The nine constructed lactic acid bacteria expression vectors were electroporated into Lactobacillus paracasei HLJ-27 competent cells to screen and obtain the nine recombinant lactic acid bacteria.Real-time quantitative PCR was used to detect the mRNA transcription level of reporter genes in recombinant bacteria,Western blotting and indirect ELISA were used to detect the protein expression,and the activity of the expressed protein was detected to evaluate the activity of the three promoters.【Result】The 720 bp of EGFP gene and the 850 bp of Ampr gene were successfully amplified by PCR.Western blotting results showed that the LUC protein of 58 ku,EGFP protein of 26 ku and Ampr protein of 28 ku were successfully expressed in the nine recombinant lactic acid bacteria.Real-time quantitative PCR and indirect ELISA results showed that the level of exogenous gene transcription and exogenous protein expression driven by the promoter Pldh were significantly higher than those of promoters PHH and Phce(P<0.05 or P<0.01).The results of reporter gene detection showed that the promoter Pldh showed higher driving activity of the foreign proteins LUC,EGFP and Ampr than the promoters PHH and Phce(P<0.05 or P<0.01).【Conclusion】Three promoter-driven reporter gene expression systems were successfully constructed,and the activity of three promoters was verified to be Pldh>PHH>Phce.Ampr reporter system was identified as a superior screening system for promoter activity screening.The results of this study provided a necessary basis for the establishment of lactic acid bacteria carrier system with high expression.