A set of Lactobacillus plantarum food-grade expression system and its application in heterologous protein expression
A technology of Lactobacillus plantarum and expression system, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve problems such as abnormal growth, rapid cell lysis and death, and achieve the effect of avoiding potential hazards of biological safety
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Embodiment 1
[0026] Bacterial strains, plasmids, main materials and reagents used and involved in the experiment of the present invention:
[0027] Lactobacillus plantarum WCFS1 was purchased from Guangdong Huankai Microbial Technology Co., Ltd.;
[0028] Food grade host Lactobacillus plantarum NZ5333;
[0029] Escherichia coli DH5α competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;
[0030] Plasmids pNZ5319 and pNZ5348 were donated by Jolanda M.Lambert of the Netherlands Dairy Research Institute;
[0031] Plasmid pSIP409 was purchased from BioVector NTCC Plasmid Vector Strain Cell Protein Antibody Gene Collection Center;
[0032] E.coli K12ΔglmS competent cells with glmS gene knockout were constructed and preserved in the laboratory;
[0033] Bacterial genome extraction kit;
[0034] SanPrep column plasmid DNA mini-extraction kit;
[0035] Ion-exchange plasmid extraction kit;
[0036] N-acetyl-D-glucosamine (GlcNAc);
[0037] Erythromycin, chloramphenic...
Embodiment 2
[0042] 1. Construction of food-grade host Lactobacillus plantarum NZ5333.
[0043] (1) Extraction of whole genome DNA of Lactobacillus plantarum WCFS1
[0044] Bacterial genome extraction kit was used to extract according to the instructions.
[0045] (2) PCR amplification of the 5' and 3' homology arms of the glmS gene and construction of the suicide plasmid pNZ5326 ⅰ. Primer design and synthesis
[0046] Using NCBI Lactobacillus plantarum WCFS1genome (GenBank: AL935263.2) as a template, the 5' and 3' homology arms of the glmS gene were designed respectively,
[0047] Homology arm primer at the 5' end of glmS gene:
[0048] A: 5'-GGG CTCGAG CAGGATACAGTCGTCACAACG-3'
[0049] B:5'-GGG ATTTAAAT CCACACATAAATTAATCTTCC-3'
[0050] Homology arm primer at the 3' end of glmS gene:
[0051] C:5'-CCC GAGCTC GTAAGTGTGTGGGCACCAAG-3'
[0052] D:5'-GGG AGATCT GATTGCCACAAGTAGCCAC-3'
[0053] ⅱ. Amplification, cloning and sequencing of 5' homology arm and 3' homology arm of glm...
Embodiment 3
[0069] The construction of the food-grade expression vector pSIP497 of embodiment 3glmS mark
[0070] 1.P ldhL Linked cloning with glmS gene fragment
[0071] i. Respectively amplify the ldhL promoter gene and the glucosamine-6-phosphate synthase coding gene glmS
[0072] The entire genome of Lactobacillus plantarum WCFS1 was extracted using a bacterial genome extraction kit, and the glmS gene sequence was retrieved with reference to the published genome sequence of Lactobacillus plantarum WCFS1 (GenBank: AL935263.2), primers were designed, and the glmS gene (1818bp) was amplified by PCR. ) and P ldhL Fragment (500bp), see Figure 4 , the primer sequences, reaction system and reaction conditions are as follows:
[0073] (i) Primer sequence:
[0074] Pldhl-F:5'-AGATCTAATTCTTCTCACCGTCTTG-3'
[0075] Pldhl-R: 5'-CACCAACAATTCCACACATTCCTACGAGAGGATGACTTATT-3'
[0076] glmSF:5'-AATAAGTCATCCTCTCGTAGGAATGTGTGGAATTGTTGGTG-3'
[0077] glmSR:5'-GGG CTCGAG TCAGTGGTGGTGGTGGTGGTGTT...
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