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Publication Number
US-10626387-B2
Publication Date
2020-04-21
Expiration Date
Abstract
The present invention discloses a lysin that is capable of killing Staphylococcus and the use thereof, belonging to the field of biological agents. The present invention discloses the amino acid sequence and the encoding gene sequence of the lysin. This lysin keeps active in a wide range of pH. It has lytic activity against Staphylococcus in pH 4-11. The recombinant protease constructed by the encoding gene can be solubly expressed in E. coli strain BL21 (DE3). The lysin can be used to effectively kill multiple species Staphylococcus in vitro, including methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) isolated in clinics. This lysin can be used as an antibiotic for the treatment of staphylococcal infections in vivo. This lysin is also able to rapidly lyse staphylococcal cell wall; as a result, intracellular substances such as ATP and DNA are released. Those released substances can be used to detect the type of Staphylococcus.
Core Innovation
The invention relates to a recombinant Staphylococcus lysin protein whose amino acid sequence is shown in SEQ.ID.NO.2. The associated gene nucleic acid sequence is shown in SEQ.ID.NO.1, linking a defined lysin identity to a defined recombinant construct. The document describes production of a soluble recombinant lysin and recovery of an active lysin preparation.
The recombinant lysin is characterized for antibacterial activity against Staphylococcus, including MSSA and MRSA. The activity is described as broad across a pH range from 4 to 11 and includes lytic effects against multiple Staphylococcus species. The document also includes experimental verification of antibacterial performance using in vivo and in vitro systems.
Beyond antibacterial killing, the document discloses workflows for rapid detection and characterization of Staphylococcus. One workflow uses ATP release with a luciferase readout to detect fluorescence intensity changes after mixing with the recombinant lysin. Another workflow uses PCR detection from lysin-lysed material, using primer sets for femB and mecA and further SCCmec strain typing using SCCmec primer sets.
Claims Coverage
The independent claim set includes one independent claim (clm-00001). The claims cover a recombinant Staphylococcus lysin protein defined by SEQ.ID.NO.2, with further inventive features for gene/DNA definition, soluble recombinant expression and purification, in vitro killing, and Staphylococcus detection/characterization using luciferase-based fluorescence and PCR/electrophoresis readouts, including MRSA determination.
Recombinant lysin with SEQ.ID.NO.2 amino acid sequence
A recombinant Staphylococcus lysin protein, wherein the amino acid sequence of said lysin is shown in SEQ.ID.NO.2.
Defined lysin gene DNA sequence as SEQ.ID.NO.1
A gene encoding a recombinant Staphylococcus lysin protein, wherein the DNA sequence of said gene is shown in SEQ.ID.NO.1.
Soluble expression and purification of the recombinant lysin
A method for preparing the soluble recombinant Staphylococcus lysin protein by cloning the gene, constructing an expression plasmid with pBAD24, expressing in E. coli BL21(DE3), lysing cells, and purifying the soluble protein by ammonium sulfate precipitation and dialysis, followed by sequential HiTrap Q Sepharose FF and HiTrap SP Sepharose FF column chromatography with gradient elution and final dialysis against PBS.
In vitro killing of Staphylococcus species by lysin mixing
A method for in vitro killing of specified Staphylococcus species by mixing an effective amount of a recombinant Staphylococcus lysin protein with a Staphylococcus bacteria suspension.
Luciferase-based detection and characterization via ATP release readout
A method that detects and characterizes Staphylococcus by mixing a Staphylococcus sample with a recombinant Staphylococcus lysin protein, adding luciferase and its substrate, incubating, and detecting fluorescence intensity changes with a microplate reader, using a sample without the recombinant lysin as a negative control.
PCR/electrophoresis characterization using femB and mecA
A method that detects and characterizes a Staphylococcus sample by mixing it with a recombinant Staphylococcus lysin protein, incubating and centrifuging to obtain supernatant, using two PCR primer pairs to amplify femB and mecA genes, and analyzing the PCR products by electrophoresis to determine whether the strain is Methicillin-resistant Staphylococcus aureus.
Overall, the claim coverage centers on a recombinant Staphylococcus lysin protein defined by SEQ.ID.NO.2, with further embodiments that add defined gene/DNA sequence, soluble recombinant expression and purification, in vitro killing by lysin mixing, and Staphylococcus detection and characterization using either luciferase-based fluorescence from ATP release or PCR/electrophoresis characterization using femB and mecA to determine methicillin resistance.
Stated Advantages
Broad anti-Staphylococcus activity across pH 4–11.
Lytic activity against MSSA and MRSA.
Documented low cytotoxicity in Caco-2 cells.
Rapid detection and typing of Staphylococcus via ATP release with luciferase readout.
Detection and characterization of methicillin resistance using PCR from ClyO-lysed supernatant DNA with femB/mecA primers.
MRSA SCCmec strain typing using SCCmec primer sets.
Documented Applications
In vivo and in vitro antibacterial treatment using the recombinant lysin against Staphylococcus, including MSSA and MRSA, verified using a scalded-mouse skin model, survival in infected mice, and other killing assays.
In vitro killing of specified Staphylococcus species by mixing an effective amount of recombinant lysin with a Staphylococcus bacteria suspension.
Rapid detection and characterization of Staphylococcus using luciferase-based fluorescence detection following mixing with the recombinant lysin.
Rapid detection and characterization of methicillin-resistant Staphylococcus aureus by PCR and electrophoresis using femB and mecA primers on DNA obtained from lysin-lysed supernatant.
MRSA SCCmec strain typing using SCCmec primer sets.
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