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High adhesion clostridium butyricum and its preparation method

A technology of Clostridium butyricum and high adhesion, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., and can solve problems such as low adhesion performance, colonization, and affecting application effects

Inactive Publication Date: 2014-07-09
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the application process of Clostridium butyricum, its low adhesion performance seriously affects its application effect, because most Clostridium butyricum cannot colonize in the intestine of animals and needs to be supplemented continuously from the outside. Mainly focus on improving the production of Clostridium butyricum, for example, the patent document with application number 201110324228.2 discloses a method for industrial production of Clostridium butyricum with ultra-high cell concentration, which greatly increases its application cost

Method used

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  • High adhesion clostridium butyricum and its preparation method
  • High adhesion clostridium butyricum and its preparation method
  • High adhesion clostridium butyricum and its preparation method

Examples

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Effect test

Embodiment 1

[0034] Example 1 Cloning of Lactobacillus Adhesin Gene Sequence

[0035] According to literature records (Complete genome sequence of Lactobacillus kefiranofaciens ZW3. Journal of Bacteriology, 2011, 193(16): 4280-4281) and NCBI (National Center for Biotechnology Information) (GeneBank accession number: NC_015602) recorded lactobacillus adhesion protein gene sequence, and according to Bacillus subtilis The multiple cloning restriction site of plasmid pHY300PLK, design gene-specific primers carrying the corresponding restriction site to amplify the target gene (Lactobacillus Adhesin gene) for the construction of recombinant expression vector.

[0036] The primers used are: 5'-CGC GGATCC ATGAATACTGTTGCTCCTC-3' (as an upstream primer, carrying B amHI restriction site) and 5'-CCG GAATTC ATTTTTTTTACGTTTTTTTT-3' (as a downstream primer, carrying E coRI restriction site, the protection bases are CGC and CCG respectively).

[0037] from Lactobacillus kefir ( Lactobacillus k...

Embodiment 2

[0042] Example 2 Construction of recombinant plasmid pHY300PLK-AP

[0043] After separation by electrophoresis, the PCR product (relative molecular weight: 271bp) of the target gene was recovered (SK8131, SanPrep Column DNA Gel Recovery Kit, Shanghai Sangong Bioengineering Company), and an appropriate amount of the recovered product was mixed with Bacillus subtilis plasmid pHY300PLK (purchased In China Plasmid Vector Strain Cell Strain Gene Collection Center) were digested with restriction endonucleases BamHI and EcoRI (restriction endonucleases were purchased from Shanghai Sangon Bioengineering Co., Ltd.), and then ligated with T4 DNA ligase (vector The build process is attached figure 2 ), transformed into DH5α, screened on the LB plate containing ampicillin and tetracycline double antibody to obtain a single clone, shake the bacteria to expand culture, and extract the plasmid. B amHI and E coRI double enzyme digestion verification (see image 3 ) and sent to Shanghai Sa...

Embodiment 3

[0044] Example 3 Transformation of Clostridium butyricum

[0045] Take the Clostridium butyricum cultured to the exponential growth phase (cultured for about 16 hours, Clostridium butyricum was isolated and preserved by our laboratory) to prepare competent cells, add the plasmid pHY300PLK-AP, mix well, and then transform the cells by electric shock , the transformed cells were cultured at 37°C, and after 1.5 hours, coated with tetracycline-containing LB medium plate, followed by anaerobic culture at 37°C for 24 hours, colony PCR was performed, and positive clones were obtained by electrophoresis detection, and false positive clones that were not successfully transformed were excluded. Positive clones were transformed Clostridium butyricum.

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Abstract

The invention discloses high adhesion clostridium butyricum and its preparation method, and belongs to the field of gene recombination. The invention provides the high adhesion clostridium butyricum and its preparation method to solve the technical problem, the main points of the technical proposal are that lactobacillus adhesion protein gene is obtained by cloning, and a lactobacillus adhesion protein gene-containing recombinant plasmid is converted into clostridium butyricum by use of a gene recombination method to obtain a clostridium butyricum recombinant strain. Through gene expression, the clostridium butyricum is capable of autologous production of a protein with the adhesion function, the adhesion ability is greatly improved, and the efficacy of the treatment of the clostridium butyricum is further improved.

Description

technical field [0001] The invention relates to the field of gene recombination, in particular to a gene recombined Clostridium butyricum and a preparation method thereof. Background technique [0002] Clostridium butyricum ( Clostridium butyricum ) is a new type of probiotic. Clostridium butyricum, also known as butyric acid bacteria, is a species in the genus Clostridium. It was first discovered and reported by Dr. Miyari Kinji of Chiba Medical University in Japan in 1933, so it is also called Miyari bacteria. In 1935, Dr. Kingi Miyairi isolated Clostridium butyricum from human feces and soil, and later found that its anaerobic cultured filtrate contained less fatty acids and had a strong intestine-regulating effect. Pathogenic bacteria, promote the growth of beneficial bacteria such as bifidobacteria and lactobacilli in the gut. Clostridium butyricum has the following effects, (1) Clostridium butyricum can effectively inhibit the disease-causing Staphylococcus, Candida...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/74C07K14/335A61K35/74A61P1/12C02F3/34C12R1/145
Inventor 孔青迟晨管斌林洪
Owner OCEAN UNIV OF CHINA
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