Lactococcus lactis food-sate secretion expression carrier and its preparing method and application

Lactococcus lactis, secreted expression technology, applied in the direction of bacterial antigen components, introduction of foreign genetic material using vectors, pharmaceutical formulations, etc., can solve the problems that expressed proteins cannot be folded correctly, exogenous protein delivery, and biological activity cannot be maintained

Active Publication Date: 2008-04-30
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The non-secretory form has many disadvantages, such as the inability to transport the synthesized foreign protein out of the cell in time, and it is easily degraded by intracellular proteases; the expressed protein cannot be folded correctly, so th

Method used

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  • Lactococcus lactis food-sate secretion expression carrier and its preparing method and application
  • Lactococcus lactis food-sate secretion expression carrier and its preparing method and application
  • Lactococcus lactis food-sate secretion expression carrier and its preparing method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the construction of secretory expression plasmid pSQ

[0078] Primers were designed according to the sequence of the published promoter P32 (SEQ ID NO: 2 and 3, the primers used in the experiment are shown in Table 1), and plasmid pMG36e (van de GM, van der Vossen JM, Kok J, et al.Construction of a lactococcal expression vector: expression of henegg white lysozyme in Lactococcus lactis subsp.lactis.Appl.Environ.Microbiol.1989; 55:224-228) is a template for amplifying the promoter fragment P32, including the -35 region and the -10 region; According to the gene sequence of Lactococcus lactis MG1363 usp45 published by GenBank (van Asseldonk, M., Rutten, G., Oteman, M., et al. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene. 1990; 95:155-160) design primer pair (SEQ ID NO:4 and 5), to Lactococcus lactis MG1363 (NCDO712, plasmad free, Gasson MJ, J.Bacteriol.1983; 154 (1): 1-9) The chromosome was used as a...

Embodiment 2

[0081] Embodiment 2, the construction of expression plasmid pSQ-nucA

[0082] 1. Cloning of the reporter gene nucA:

[0083] Primers (SEQ ID NO: 6 and 7), the nucA gene sequence was amplified by PCR using the chromosome of Staphylococcus aureus 25923 (purchased from China National Institute of Pharmaceutical and Biological Products) as a template. The amplified product is the coding sequence of the endonuclease mature peptide, excluding the secretory signal peptide coding sequence, and the expected length of the amplified product is about 510bp. After the amplified product was recovered and purified, it was sequenced by T-A clone and compared with the published sequence.

[0084] 2. Construction of expression plasmid pSQ-nucA:

[0085] The amplified fragment of the nucA gene with correct sequencing was double digested with Xba I and Kpn I, and ligated with the same double digested pSQ plasmid. The ligation product was transformed into competent cells E.coli X51, and the pl...

Embodiment 3

[0088] Embodiment 3, construction of secretory expression plasmid pSQZ-nucA

[0089] Primers (SEQ ID NO: 8 and 9) were designed according to the L.lactis MG1363 usp45 gene sequence (GenBank NC009004) published by GenBank, and the L.lactis MG1363 chromosome was used as a template to amplify the usp45-related sequence, including the usp45 promoter (-35 , -10 region), ribosome binding site (RBS), signal peptide sequence (SPusp45) and the first 11 amino acid coding sequences of usp45 mature peptide. The expected length of PCR amplified fragment is 230bp.

[0090] According to a method similar to that of Examples 1-2, the target gene was cloned and sequenced by T-A, and it was confirmed that it was completely consistent with the related sequence of usp45 published on GenBank. After the clones with correct sequencing were digested with Xba I and HindIII, the small fragments were recovered from the gel, connected with the pSH91 plasmid of the same digestion, and transformed into E.c...

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Abstract

The invention relates to lactic acid galactococcus food-grade secretion expression vector, and the preparation method and the application thereof, in particular to the lactic acid galactococcus food-grade secretion expression vector which comprises the following components: a thyA gene selection marker, replicons of plasmid pWV01, multiple cloning sites, promoter used for secretion expression, a ribosome bind site of Usp45, a signal peptide sequence of Usp45 and a partial Usp45 mature peptides coded sequence.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a secreted expression carrier capable of secreting and expressing foreign genes in Lactococcus lactis and its preparation method and application. Background technique [0002] Food-grade expression systems are gaining increasing attention due to their safety and compliance with FDA requirements. The food-grade expression system must meet the following conditions: (1) The vector must be food-grade and must not contain non-food-grade functional DNA fragments, such as antibiotic resistance genes, etc.; (2) The expression host must be safe, with clear and stable characteristics Food-grade microorganisms (generally regarded as safe, GRAS), such as Lactococcus lactis, Lactobacillus acidophilus, Bifidobacterium and other strains that have been widely used in the food industry for a long time. In addition, the host bacteria of a food-grade system must be sufficiently stable in food and aft...

Claims

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Application Information

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IPC IPC(8): C12N15/74A61K39/02A61K48/00
Inventor 孙强正徐建国
Owner ICDC CHINA CDC
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