Preparation and application of mycobacteriophage lyase Lysin-Guo1

Abstract

The invention relates to the technical field of molecular biology, in particular to preparation and application of mycobacteriophage lyase Lysin-Guo1. The invention provides the preparation and application of the mycobacteriophage lyase. The invention provides a bacteriostatic agent; the bacteriostatic agent is prepared from main active components selected from at least one of the mycobacteriophage lyase Lysin-Guo1, a carrier containing a Lysin-Guo1 expression element, an expression cassette containing a Lysin-Guo1 expression element, or host cells containing Lysin-Guo1 expression elements; the Lysin-Guo1 has an amino acid sequence shown in SEQ ID No 1. The invention also provides a preparation method of the mycobacteriophage lyase Lysin-Guo1. The mycobacteriophage lyase Lysin-Guo1 is capable of cleaving multiple types of mycobacterium tuberculosis, and is also found to have a certain inhibiting effect on other bacteria including escherichia coli, staphylococcus aureus, acinetobacter baumannii and pseudomonas aeruginosa. The preparation and application of the mycobacteriophage lyase Lysin-Guo1 lay a foundation for anti-tuberculosis substitution therapy of drug-resistant tuberculousbacillus.

Description

technical field

[0001] The invention relates to the technical field of molecular biology, in particular to the preparation and application of mycobacteriophage lyase Lysin-Guo1. Background technique

[0002] There are many types of Mycobacterium, which can be divided into three types: Mycobacterium tuberculosis complex, non-tuberculous mycobacteria and Mycobacterium leprae.

[0003] Mycobacterium tuberculosis (M.tuberculosis), commonly known as Mycobacterium tuberculosis, is an obligate aerobic, Gram-positive bacterium. It is a slender and slightly curved bacillus with a size of 1-4*0.4μm. In recent years, it has been found that Mycobacterium tuberculosis has a capsule outside the cell wall, and the capsule has a certain protective effect on Mycobacterium tuberculosis. Mycobacterium tuberculosis does not produce endotoxins and exotoxins. Its pathogenicity may be related to the inflammation caused by the massive reproduction of bacteria in tissue cells, the toxicity of bact...

Examples

Embodiment 1

[0053] Example 1 Isolation and Identification of Mycobacteriophage Guo1

[0054] 1. Isolation of mycobacteriophage

[0055] Using Mycobacterium smegmatis as an indicator bacterium, isolate phages from the flower pot soil in the tuberculosis ward of a hospital, the specific operation steps are as follows:

[0056] 1. Specimen treatment: Take 5g of soil and soak it in 10mL of phage buffer for 30min. Let the phage fully enter the buffer, centrifuge at 4500g for 10min, carefully absorb the supernatant, filter and sterilize with a 0.22μm filter.

[0057] 2. Phage amplification:

[0058] 1) Take 1 mL of the above supernatant and add 4 mL of Mycobacterium smegmatis cultivated to the logarithmic growth phase, mix well and let stand at room temperature for 15 minutes to allow the mycobacteriophage to adsorb Mycobacterium smegmatis.

[0059] 2) Add the mixed solution to 10mL 7H9 liquid medium, culture overnight at 37°C with 160rpm shaking on a shaker.

[0060] 3) Collect the culture...

Embodiment 2

[0104] Embodiment 2, expression and purification of Lysin-Guo1 protein

[0105] 1. Gene amplification and recombinant construction

[0106] 1. Acquisition of Lysin-Guo1 gene

[0107] Using the genomic DNA of phage Guo1 as a template, PCR amplification was performed using primers 1 and 2. The reaction system is as follows: Guo1 genomic DNA 0.5 μL, 5×Buffer 10 μL, dNTPs 4 μL, Taq enzyme 0.5 μL, primer 1 2 μL, primer 2 2 μL, deionized water to make up to 50 μL. Reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 1 min.

[0108] Primer 1: 5'- CGCCATATGG TCAGAACACCAGCCCGAGT-3' (the underlined part is the recognition site of NdeI, and the sequence thereafter is the 1st-19th position of SEQ ID NO.2); Primer 2: 5'- CCGCTCGAG ATGGCCGATCGTTTCTTCC-3' (the underlined part is the recognition site of XhoI, and the sequence thereafter is the reverse complementar...

technical field

[0001] The invention relates to the technical field of molecular biology, in particular to the preparation and application of mycobacteriophage lyase Lysin-Guo1. Background technique

[0002] There are many types of Mycobacterium, which can be divided into three types: Mycobacterium tuberculosis complex, non-tuberculous mycobacteria and Mycobacterium leprae.

[0003] Mycobacterium tuberculosis (M.tuberculosis), commonly known as Mycobacterium tuberculosis, is an obligate aerobic, Gram-positive bacterium. It is a slender and slightly curved bacillus with a size of 1-4*0.4μm. In recent years, it has been found that Mycobacterium tuberculosis has a capsule outside the cell wall, and the capsule has a certain protective effect on Mycobacterium tuberculosis. Mycobacterium tuberculosis does not produce endotoxins and exotoxins. Its pathogenicity may be related to the inflammation caused by the massive reproduction of bacteria in tissue cells, the toxicity of bact...

Examples

Embodiment 1

[0053] Example 1 Isolation and Identification of Mycobacteriophage Guo1

[0054] 1. Isolation of mycobacteriophage

[0055] Using Mycobacterium smegmatis as an indicator bacterium, isolate phages from the flower pot soil in the tuberculosis ward of a hospital, the specific operation steps are as follows:

[0056] 1. Specimen treatment: Take 5g of soil and soak it in 10mL of phage buffer for 30min. Let the phage fully enter the buffer, centrifuge at 4500g for 10min, carefully absorb the supernatant, filter and sterilize with a 0.22μm filter.

[0057] 2. Phage amplification:

[0058] 1) Take 1 mL of the above supernatant and add 4 mL of Mycobacterium smegmatis cultivated to the logarithmic growth phase, mix well and let stand at room temperature for 15 minutes to allow the mycobacteriophage to adsorb Mycobacterium smegmatis.

[0059] 2) Add the mixed solution to 10mL 7H9 liquid medium, culture overnight at 37°C with 160rpm shaking on a shaker.

[0060] 3) Collect the culture...

Embodiment 2

[0104] Embodiment 2, expression and purification of Lysin-Guo1 protein

[0105] 1. Gene amplification and recombinant construction

[0106] 1. Acquisition of Lysin-Guo1 gene

[0107] Using the genomic DNA of phage Guo1 as a template, PCR amplification was performed using primers 1 and 2. The reaction system is as follows: Guo1 genomic DNA 0.5 μL, 5×Buffer 10 μL, dNTPs 4 μL, Taq enzyme 0.5 μL, primer 1 2 μL, primer 2 2 μL, deionized water to make up to 50 μL. Reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 1 min.

[0108] Primer 1: 5'- CGCCATATGG TCAGAACACCAGCCCGAGT-3' (the underlined part is the recognition site of NdeI, and the sequence thereafter is the 1st-19th position of SEQ ID NO.2); Primer 2: 5'- CCGCTCGAG ATGGCCGATCGTTTCTTCC-3' (the underlined part is the recognition site of XhoI, and the sequence thereafter is the reverse complementar...

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