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A recombinant Bacillus subtilis engineering bacterium producing pig glutamine synthetase, its construction method and its application

A technology of Bacillus subtilis and porcine glutamine, applied in the field of animal genetic engineering, can solve problems such as easy decomposition, instability, and limited application

Active Publication Date: 2021-06-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low solubility of glutamine, it is very unstable in water, easy to decompose and other characteristics, as well as the expensive international market price, etc., which limit its application in animal husbandry

Method used

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  • A recombinant Bacillus subtilis engineering bacterium producing pig glutamine synthetase, its construction method and its application
  • A recombinant Bacillus subtilis engineering bacterium producing pig glutamine synthetase, its construction method and its application
  • A recombinant Bacillus subtilis engineering bacterium producing pig glutamine synthetase, its construction method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of recombinant Bacillus subtilis engineering bacteria producing pig glutamine synthetase

[0048] In this example, the starting plasmid PDG1730 and wild-type Bacillus subtilis WB800 were donated by Professor Ming Sun of the School of Life Science and Technology of Huazhong Agricultural University, and Escherichia coli DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0049] S1: Design primers for P43 gene and SacB gene respectively, and amplify to obtain corresponding gene fragments.

[0050] According to the fragment sequence of the P43 gene (promoter) in NCBI (accession number NCBI-Gene ID: EF473728.1) and the sequence of the signal peptide SacB gene fragment (accession number NCBI-Gene ID: MH614588.1), the primers were designed as follows:

[0051] Forward primer P43-F: CGggatccgagctcagctttatgagtgg, see SEQ ID NO: 1;

[0052] Reverse primer P43-R: GCAAACTTTTTGATGTTCATgtgtacattcctctcttacc, see SEQ ID NO: 2;

[0053] Forward ...

Embodiment 2

[0082] Example 2 Secretion and expression of pGS by gene recombinant Bacillus subtilis

[0083] The verified correct recombinant Bacillus subtilis strain RBs-GS will be fermented.

[0084] The specific steps are: pick a single clone and inoculate it in 5ml of LB medium, activate it at 37°C and 200r / min for 12-24h; take the activated bacterial solution and inoculate it in 50mL In the LB medium of 37°C, cultivated at 200r / min for 2-14h, take the fermentation suspension of the recombinant bacteria and the fermentation suspension of the control bacteria (ie wild-type Bacillus subtilis WB800), and use Shanghai Enzyme The GSElisa assay kit produced by Science and Technology Co., Ltd. detects the content and activity of the fermented bacteria suspension (i.e., the pGS obtained) according to the kit operation manual, and obtains the following results: Figure 9 with Figure 10 The results shown.

Embodiment 3

[0085] Example 3 The protective effect of recombinant Bacillus subtilis RBs-GS on the intestinal tract of offspring rats

[0086] The recombinant Bacillus subtilis strain RBs-GS of the present invention is used as a probiotic preparation, and the preparation method is as follows: RBs-GS is cultured in a shaker at 37° C. and 200 r / min for 6 hours to obtain a fermented bacterial suspension that is a probiotic preparation. The purpose of feeding weaned rats with damaged intestines is to verify the ability of the recombinant Bacillus subtilis RBs-GS of the present invention to repair the damaged intestines of weaned rats at the animal level.

[0087] Test grouping

[0088] For the animal experiment, 200 weaned pups with similar age and weight at 19-21 days were selected and divided into 10 groups, with 3 repetitions in each group. The test period was 15 days. The basic diet was provided by the Animal Experiment Center of Huazhong Agricultural University as a routine The basal die...

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Abstract

The invention belongs to the technical field of animal genetic engineering, and in particular relates to a recombinant Bacillus subtilis engineering bacterium producing pig glutamine synthetase, a construction method and application thereof. Using Bacillus subtilis as the starting strain, the amylase gene amy‑E was knocked out through homologous recombination and glutamine synthetase (GS) from porcine was introduced to construct a stable and high-secretion engineered bacterium, realizing porcine glutamine synthetase efficient secretory expression. Animal experiments show that the strain can prevent and treat diarrhea in animals, increase the secretion of anti-inflammatory factor IL-10, and at the same time reduce the secretion of inflammatory factors IFN-γ, IL-1β, TNF-α, etc.; repair damaged intestines, promote Intestinal villi grow, ultimately improving animal performance. It lays the foundation for the industrial production of pig-derived glutamine synthetase, and provides technical support for the substitution of antibiotics in feed and the healthy and sustainable development of animal husbandry.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to a recombinant Bacillus subtilis engineering bacterium producing pig glutamine synthetase, a construction method and application thereof. Background technique [0002] Under the modern intensive breeding production system, in order to obtain the greatest economic benefits, middle and early weaning of piglets is widely used. Weaning is the biggest stress that piglets face after birth. Due to the imperfect physical function of piglets at weaning, the short-term decline in feed intake and the impact of adverse external environments, it is very easy to cause imbalance and dysfunction in the intestinal micro-ecological environment of piglets 1. Intestinal development is blocked, resulting in decreased production performance, stagnant growth and development, and susceptibility to pathogenic microorganisms. With the advance of weaning age, this weaning str...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12P13/14A23K10/18C12R1/125
CPCA23K10/18C12N9/93C12N15/75C12P13/14C12Y603/01002
Inventor 王喜亮李越石德时李毕平贺宇成许青荣
Owner HUAZHONG AGRI UNIV
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