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

doi:10.16431/j.cnki.1671-7236.2023.11.006

Abstract

Abstract: 【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.

Key words: lactic acid bacteria expression system; constitutive promoter; reporter gene system; expression of foreign protein

CLC Number:

S816.3

YUAN Shihao, ZHANG Hailin, JU Ning, WANG Xinle, ZHAO Haiyuan, SHAO Yilan, LI Jiaxuan, JIANG Yanping, CUI Wen, TANG Lijie, LI Yijing, WANG Xiaona. Comparison of the Effect of Different Constitutive Promoters on Exogenous Gene Expression in Lactobacillus Expression System[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4370-4380.

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

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