Construction Of A Food-grade Expression Vector For Lactic Acid Bacteria

Most lactic acid bacteria (LAB) are considered as safe probiotics. Food-grade genemodified LAB and their expression products can be directly used in food, pharmaceutical andhealth care industries. The prospect of constructing food-grade LAB vectors for expressingsome useful protein is considered to be quite attractive. In this paper, nisin resistance gene(NSR) was amplified from nisin-resistant LABs as a food-grade selection marker in place oferythromycin resistance marker of Lactococcus lactis vector pMG36e, so as to construct aLAB food-grade vector pMG36n, and then pMG36n was transformed into LAB byelectroporation.5nisin resistant strains, named N1, N2, N3, N4, N5respectively, were initially screenedfrom fresh milk using medium containing nisin and bromocresol purple. LABs preserved byour laboratory were also selected, and it was found that Lactococcus lactis subsp. lactis andM2(Streptococcus thermophilus) were sensitive to nisin and did not contain plasmid as well.Therefore, the two strains could be used as host strains after building a vector with NSR as aselection marker.Genomes and plasmid of N1, N2, N3, N4, N5was used respectively as templates toamplify NSR conserved sequence, a fragments about400bp was obtained from plasmids ofN1, N2, N5separately, which also indicated that NSR gene was located in the plasmid but notin the genome. Physiological and biochemical tests as well as Lactococcus lactis specific16S rDNA sequence analysis revealed that N1, N2, N5all belonged to Lactococcus lactissubsp. lactis. The fermentation broth of the above3strains showed non-inhibition toStaphylococcus aureus. That was to say those3strains were not nisin-producing strains.16S rDNA sequencing results of the3strains was submitted to the GenBank, and sequencenumbers were HQ647114, HQ647115, HQ647116, respectively.NSR full-length fragments about1000bp, including the start codon and transcriptionterminator were obtained by PCR, using plasmids of N1, N2, N5as templates. NSR gene ofN1was connected to vector pMD19-T and sequenced, sequence number of which inGenBank was HQ701130. Bioinformatics analysis revealed that, the full length of NSR ofN1was1014bp, containing383Adenine(A),149Cytosine(C),175Guanine(G) and307Thymine(T). Open reading frame (ORF) was between4to960bases, coding for318amino acids. There was a significant hydrophobic region at the position of the12th amino acids, and a trans-membrane structure might exist between the10th and30th amino acids.NSR gene of N1was inserted into the multiple cloning restriction sites of vectorpMG36e, and transformed into E. coli DH5α by CaCl2method. Results showed that, thetransformed NSR gene could make the host bacteria resistant to nisin, and thus could be usedas a selection marker.Gene fragment of vector pMG36e apart from erythromycin resistance gene wasamplified by PCR and named pMG36e-. NSR of N1and pMG36e-fragment wereconnected, for the purpose of constructing a food-grade vector pMG36n. pMG36n was thensuccessfully transformed into Lactococcus lactis subsp. lactis by electroporation, theoperation parameters of the process was as follows: voltage2000V, capacitor25μF, pulsetime5ms. Furthermore, the transformant strain could grow on the selective mediumsupplemented with nisin.