关键词: 隐蔽性质粒, 滚环复制, 穿梭质粒, 植物乳杆菌

Abstract: Objective: In order to construct an expression vector for Lactobacillus plantarum, a plasmid carried by the strain was subjected to sequence determination and functional analysis, and an expression system was constructed by using the plasmid system. Through this study, we aim to make a positive contribution to the preparation of oral lactic acid bacterial vaccines. Methods: The plasmid (pLP3) was extracted from L. plantarum LP3, sequenced and subjected to functional analysis. An Escherichia coli/Lactobacillus shuttle vector was constructed based on the replicon of pLP3, the chloramphenicol resistance gene from pSCPSP as a selection marker and the replicon of pUC19. Furthermore, an expression vector D-pLP3-PslpA was developed from the shuttle vector incorporated with the promoter PslpA of S-layer protein from Lactobacillus acidophilus and the gene green fluorescent protein (eGFP) as a reporter gene in L. plantarum. Results: pLP3 was a cryptic plasmid in L. plantarum LP3, isolated from traditional fermented sauerkraut. The size of pLP3 was 2 017 bp and its GC content was 37.48%. Based on the backbone of pLP3, we successfully constructed the shuttle vector D-pLP3 and electroporated it into several strains of lactic acid bacteria. The transformation efficiencies ranged from 0.3 × 102 to 1.0 × 103 CFU/μg DNA. eGFP was expressed successfully in L. plantarum. Conclusion: The expression vector D-pLP3-PslpA possesses the potential to be used as a molecular tool for heterologous gene cloning and expression in Lactobacillus.

Key words: cryptic plasmid, rolling circle replication, shuttle vector, Lactobacillus plantarum