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Genetically engineered bacterium for producing resuscitation promoting factors and application of genetically engineered bacterium

A technology of genetically engineered bacteria and factors, applied in the field of bioengineering, to achieve the effect of increasing the separation abundance, simple operation, and short cultivation period

Inactive Publication Date: 2017-07-21
ENVIRONMENTAL SCI RES & DESIGN INST OF ZHEJIANG PROVINCE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on RPF mainly focuses on the recovery of human VBNC bacteria (such as Mycobacterium tuberculosis), immunology and rapid diagnosis of tuberculosis. There are few reports on the discovery of microbial resources at home and abroad

Method used

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  • Genetically engineered bacterium for producing resuscitation promoting factors and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing resuscitation promoting factors and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing resuscitation promoting factors and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Cloning of recovery-promoting factor genes and construction of genetically engineered bacteria

[0031] (1) Culture of Micrococcus luteus and extraction of genomic DNA

[0032] Micrococcus luteus was inoculated into lactate minimal medium (Lactate Minimal Medium, LMM) for revival culture at 35°C.

[0033] LMM medium formula: 4.0g NH 4 Cl, 1.4g KH 2 PO 4 , 0.005g Biotin, 0.02g L-Methionine, 0.04g Vitamin B1, 1.0g Inosine, 0.03g MgSO 4 , 10.0g L-lactate lithium salt (Lithium L-lactate) and 1.0mL mineral salt solution (0.375g CuSO 4 ·5H 2 O, 0.785g MnCl 2 4H 2 O, 0.183g FeSO 4 ·7H 2 O, 0.029g Na 2 MoO 4 2H 2 O and 0.089g ZnSO 4 ·7H 2 0), add distilled water 1000mL to dissolve, adjust the pH to 7.5, and sterilize under high temperature and high pressure.

[0034] Take 5mL Micrococcus luteus culture solution and collect the cells by centrifugation, resuspend the pellet with 200μL of 25mmol / L Tris-HCl buffer (pH8.0, containing 50mmol / L glucose, 10mmol / ...

Embodiment 2

[0043] Example 2: Construction, expression and purification of recovery-promoting factor genetically engineered bacteria

[0044] Inoculate the Escherichia coli containing the recombinant plasmid into 5 mL of LB medium (containing 50 μg / mL kanamycin) and culture overnight at 37°C with shaking; the next day, inoculate it into 200 mL of fresh medium with the same resistance, and culture it with shaking at 37°C. body growth to OD 600 When the temperature is 0.6-0.8, add 100mM isopropylthioβ-D-galactoside (IPTG) to a final concentration of 1mM, lower the culture temperature to 22°C, and induce the bacteria to express a large amount of target protein. To avoid the formation of inclusion bodies, collect the cells by centrifugation after culturing at 100 rpm for 12-16 hours.

[0045] Add 5-10 times the volume of 50mM Tris-HCl buffer (pH8.0) to resuspend the bacteria, ultrasonically disrupt for 10 minutes; centrifuge (12000rpm, 30min), collect the supernatant to obtain a crude extrac...

Embodiment 3

[0048] Embodiment 3: the revival-promoting growth-promoting effect of revival-promoting factors on microorganisms in soil samples

[0049] (1) Prepare beef extract peptone bacterial culture medium: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, and equal volumes are packed in micro culture bottles (Azowon, Japan, article number: 2-867-01), 10mL / bottle, high temperature and high pressure sterilization.

[0050] (2) Source of soil samples: Soil samples collected from the leachate homogenization pool of Tianziling landfill in Hangzhou.

[0051] (3) In the ultra-clean workbench, take 100 mg of soil samples and place them in a sterilized 1.5 mL centrifuge tube, add 1 mL of sterilized deionized water, shake and mix well, and centrifuge to collect the supernatant; take out the purified and frozen RPF protein and place in Use after the ice bath has melted.

[0052] (4) MPN (Most Probable Number) counting method culture: the experiment was divided into two groups (RPF treatment group a...

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Abstract

The invention discloses a genetically engineered bacterium for producing resuscitation promoting factors and application of the genetically engineered bacterium. The classified name of the genetically engineered bacterium for producing the resuscitation promoting factors is Escherichia Coli, the bacterial strain number is pET28a-RPF(WT), and the collection number is CGMCC No.13217. The genetically engineered bacterium is collected in China General Microbiological Culture Collection Center of Institute of Microbiology of Chinese Academy of Science, which is located at No.3 of first yard of Beicheng West Road of Chaoyang District of Beijing City, in October 31 of 2016. The genetically engineered bacterium has the advantages that growth of potential dormant bacteria in soil samples is promoted by RPF albumen made from the engineered bacterium, separation abundance is improved, and the genetically engineered bacterium can be applied to exploration and separation of microbial resources in a viable but non-culture (VBNC) state in environmental samples (soil, water, etc.).

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a genetically engineered bacterium for producing resuscitation promoting factor (RPF) and a method for rapidly preparing high-purity RPF protein using the genetically engineered bacterium, in particular to the application of recombinant proteins. Background technique [0002] The resuscitation promoting factor (Resuscitation Promoting Factor, RPF) is discovered by the Russian Mukamolova research group and is secreted by Micrococcus luteus in the late logarithmic growth phase. -12 mol / L) concentration can make the self-bacteria in a viable but non-culturable (VibleBut non-Culturable, VBNC) state recover its growth and reproduction ability. In addition to promoting the recovery of its own VBNC bacteria, RPF can also recover some species of Gram-positive bacteria Mycobacterium that are close to it in the VBNC period. Genes similar to this rpf are also found in Streptomyces,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C07K14/305C12N1/38C12N1/20C12R1/19C12R1/07C12R1/01
CPCC07K14/305C12N1/20C12N1/38
Inventor 张宇丁林贤李明智梅荣武王慧荣李思亮任旭锋苏晓梅
Owner ENVIRONMENTAL SCI RES & DESIGN INST OF ZHEJIANG PROVINCE
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