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Lyase capable of killing streptococcus equi subspecies. Equi and medical application of lyase

A technology of streptococcus equi and lyase, applied in the field of bioengineering

Pending Publication Date: 2021-03-16
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, there are few reports on the lyase of Streptococcus equi subspecies equi

Method used

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  • Lyase capable of killing streptococcus equi subspecies. Equi and medical application of lyase
  • Lyase capable of killing streptococcus equi subspecies. Equi and medical application of lyase
  • Lyase capable of killing streptococcus equi subspecies. Equi and medical application of lyase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Expression and purification of lyase

[0030] The primers for amplifying the lyase LysLF1 are:

[0031] Upstream 5'-CGGGATCCATGGCTACATATCAGGAATATAAAAGTC-3';

[0032] Downstream 5'-CGCTCGAGTTATACTTGTGTTGCATTAGAT-3';

[0033] Pass the amplified LysLF1 gene through the specific enzyme cutting site xho I and Bam H I was connected to the Pet28a vector to construct the expression vector pET28a-LysLF1. Transform the constructed vector into Escherichia coli BL21 competent, amplify in 500 mL of fresh LB medium, add 1 / 1000 kanamycin sulfate at the same time, and wait until the bacterial solution OD 600 When the value reached 0.6-0.8, 1 / 1000 IPTG (final concentration 1 mM) was added for induction, the induction temperature was 16°C, and the induction time was 16 h.

[0034] Use Ni-NTA to purify the protein by affinity chromatography: collect the induced bacterial solution (4°C, 10000 × g, 20min), resuspend it with an appropriate amount of Tris-Cl buffer (pH=7.5), and take ...

Embodiment 2

[0042] Determination of Antibacterial Ability of Lyase LysLF1 Solid Plate

[0043] Streptococcus equi subsp. equi 3518 in the logarithmic growth phase was evenly spread on the BHI solid medium plate in the ultra-clean bench, and after drying, 10 µL of the purified lyase LysLF1 was dropped on the plate and marked. In addition, 10 µL of Tris-Cl buffer was dropped on the plate as a control. Place the plate in a 37°C incubator for overnight culture, and observe the formation of the inhibition zone the next day.

[0044] The result is as figure 2 As shown, clear plaques appeared at the markers, indicating that the lyase LysLF1 has lytic activity against Streptococcus equi subsp. equi.

Embodiment 3

[0046] Determination of Bactericidal Activity of Lyase LysLF1 in Vitro

[0047] Streptococcus equi subsp. 600 Adjust the value to around 0.6. Positive control set two groups, LysLF1 treatment group added a single dose of LysLF1 with a final concentration of 100 µg / mL. The LysLF1* treatment group was added with lyase LysLF1 at a final concentration of 100 μg / mL every 10 min, and added 3 times continuously. An equal volume of Tris-Cl buffer was added to the negative control group. The three groups were incubated in a 37°C water bath at the same time, samples were taken every 10 minutes, and colonies were counted by doubling dilution. The experiment was carried out for 60 minutes and repeated three times.

[0048] The result is as image 3 As shown, the number of colonies in the negative control group remained basically unchanged after 60 min, and the number of colonies in the single-dose LysLF1 and multiple-dose LysLF1 treatment groups decreased significantly, and the multi-...

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Abstract

The invention provides lyase capable of killing streptococcus equi subspecies. Equi and medical application, and provides a new bacteriophage lyase which can be independently used or compounded with other substances for use, has strong lytic activity and a wide lytic spectrum, can effectively kill streptococcus equi subsp. Equi and streptococcus suis. The invention provides a novel medicine for preventing and treating diseases caused by streptococcus equi subsp. Equi and streptococcus suis infection.

Description

technical field [0001] The invention discloses a Streptococcus equi subsp. equi lyase LysLF1, which has bactericidal activity on both Streptococcus equi subsp. equi and Streptococcus suis and has a wide cracking spectrum, belonging to the technical field of bioengineering. Background technique [0002] Streptococcus equi subspecies equi (S.equi) is a Gram-positive bacterium, observed under an electron microscope, most of them exist in pairs or chains, and individual bacteria are round or oval. After Gram staining, it was observed to be purple, and 5-10 bacteria formed a long chain. The long chain formed by the bacteria cells was at right angles to its long axis. When nutrients were scarce, the bacteria would exist in pairs. In addition, the bacterium cannot form spores, does not have motility, and will form capsules under special circumstances. The host of Streptococcus equi subspecies equine is an animal of the genus Equus. It is traditionally believed that it mainly infec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21A61K38/51A61P31/04C12R1/19
CPCC12N9/88C12N15/70A61P31/04A61K38/00
Inventor 顾敬敏韩文瑜冀亚路董建宝朱伟
Owner JILIN UNIV
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