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alr (alanine racemase)-deficient lactobacillus plantarum NC8

A technology of Lactobacillus plantarum and ems-alr-emx, applied in plant genetic improvement, botany equipment and methods, bacteria, etc., can solve problems such as biological safety hazards, affecting gene expression, and application value limitations

Inactive Publication Date: 2016-07-06
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] pCJ1 can replicate in Escherichia coli and express foreign proteins in Lactobacillus plantarum, but the plasmid vector uses the erythromycin resistance gene (erm) as a selection marker, once the resistance gene is transferred to the host chromosome, it is very difficult Serious biological safety hazards may arise, so the potential application value of the system is limited
In addition, Nguyen (Nguyen, et al, 2011) believed that erythromycin would be degraded under acidic conditions, which would adversely affect the induced expression of foreign genes
Erythromycin can also cause "physiological stress" in the bacteria, thereby affecting the expression of target genes

Method used

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  • alr (alanine racemase)-deficient lactobacillus plantarum NC8
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  • alr (alanine racemase)-deficient lactobacillus plantarum NC8

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Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Construction of alanine racemase gene alr deletion suicide plasmid pNZ5319-Δalr

[0038] 1. Main experimental materials and molecular biology reagents

[0039] (1) Strains and plasmid vectors

[0040] Lactobacillus plantarum NC8 strain was donated by senior researcher Anbazhagan.K of Kamaraj University in India; Escherichia coli JM109 was preserved by our laboratory; plasmids pNZ5319 and pNZ5348 were donated by Dr. Ingridvan Alen of NIZOfoodresearch; pET41a vector was provided by Novagen; pCJ1 (pSIP409), preserved by our laboratory.

[0041] (2) Main materials and reagents

[0042] Gram-positive bacterial genome extraction kit, plasmid mini-extraction kit, gel recovery kit, PCR product recovery kit are provided by Beijing Tiangen Biotechnology Company; high-fidelity PCR polymerase, restriction endonuclease, T4DNA ligase Provided by Takara Company; electric cup provided by Bio-Rad Company; erythromycin and chloramphenicol provided by Sigma; other main reage...

Embodiment 2

[0099] Example 2 Construction of alanine racemase gene alr marker plasmid expression vector pCJ2

[0100] In order to replace the erythromycin marker gene on the pCJ1 vector without mutation, the 343 bp of the upper arm end of the erythromycin gene (including the restriction site BamHI) on the pCJ1 vector, the 1128 bp of the alr gene, and the erythromycin gene on the pCJ1 vector were respectively amplified by PCR. The 567bp fragment at the downstream arm end of the gene (including the enzyme cutting site ClaI), after the PCR product was recovered and purified, the EmSF and EmXR primers were added and the three fragments were connected by SOE-PCR single-tube reaction to obtain the EmS-alr-EmX fragment with a base sequence of 2038bp . After subcloning, the linked fragment was double-digested with BamHI and ClaI, and the digested product was recovered, purified and quantified, linked with the pCJ1 fragment double-digested with the same enzyme, and transformed into the alanine-defic...

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Abstract

The invention discloses alr (alanine racemase)-deficient lactobacillus plantarum NC8 and an alr-marked expression vector pCJ2. The alr-deficient lactobacillus plantarum NC8 is alr-gene-deficient lactobacillus plantarum NC8 obtained after transfecting an alr-deficient suicide plasmid pNZ5319-Deltaalr with lactobacillus plantarum NC8, and in addition, the alr-marked expression vector pCJ2 is constructed. A food-grade lactobacillus expression system is implemented, and safety and reliability are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to alanine racemase gene (alr) deficient plant lactobacillus NC8 and alr marker expression vector pCJ2. Background technique [0002] Lactobacillus plantarum (Lactobacillus plantarum) is a common lactic acid bacteria and has a wide range of applications in industrial lactic acid fermentation and medical care. Studies have shown that Lactobacillus plantarum usually colonizes the digestive tract of humans and animals and exerts a beneficial effect (de Vries, et al, 2006). Microecological preparations made from this strain can enhance the body's immunity and effectively prevent some gastrointestinal diseases. the occurrence of disease. At the same time, this strain is also a good exogenous protein expression system. Using it as an oral pharmaceutical protein carrier largely solves the problem that proteins are easily hydrolyzed in the digestive tract, so that a variety of oral...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N15/10C12N15/75C12N1/21C12N15/65C12R1/25
CPCC12N9/90C12N15/10C12N15/65C12N15/75C12N2800/101C12Y501/01001
Inventor 王春凤姜延龙杨桂连刘琼蔡若鹏
Owner JILIN AGRICULTURAL UNIV
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