Chimeric enzyme antibiotic for killing staphylococcus aureus and preparation and application thereof

A technique for staphylococcus and staphylococcus infection, applied in biochemical equipment and methods, preparations for in vivo tests, medical preparations containing active ingredients, etc., can solve problems such as no good therapeutic effect

Inactive Publication Date: 2019-04-23
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a class of macromolecular proteases, antibiotics of this type of enzymes cannot freely enter the interior of eukaryotic cells, so they do not have a good therapeutic effect on intracellular infections caused by bacteria

Method used

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  • Chimeric enzyme antibiotic for killing staphylococcus aureus and preparation and application thereof
  • Chimeric enzyme antibiotic for killing staphylococcus aureus and preparation and application thereof
  • Chimeric enzyme antibiotic for killing staphylococcus aureus and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 designs primers, PCR amplifies target gene fragment

[0048] Referring to the genome sequences of Staphylococcus aureus phage, cytoplasmic transduction peptide and green fluorescent protein GFP in GenBank, the lyase JDlys gene, three transmembrane peptides (CTP Tat 、CTP ANT and CTP TP10 ) gene and green fluorescent protein GFP gene; design primers, use the synthesized gene as a template, and use PCR technology to amplify the target gene, wherein the size of JDlys gene is 1488bp, CTP Tat The gene size is 33bp, CTP ANT Gene size is 48bp, CTP TP10 The gene size is 63bp, and the GFP gene size is 720bp. The specific operation is as follows:

[0049] Primers were designed, and the primer sequences are listed in Table 1. Using the DNA of synthetic lyase JDlys gene, transmembrane peptide CTP gene and green fluorescent protein GFP as templates, the JDlys gene fragment was amplified with primers JDlys-F1 / JDlys-R; primers CTP Tat -F / CTP Tat -R, CTP Ant -F / C...

Embodiment 2

[0052] Example 2 Construction, expression and purification of recombinant plasmid pET-28a(+)-CTP-JDlys

[0053] Using the prokaryotic expression system pET-28a(+), the JDlys gene product obtained in Example 1 was used to construct a recombinant plasmid to obtain the recombinant plasmid pET-28a-JDLys, and then the 5' end of the JDlys gene in pET-28a-JDlys was digested, Insert CTPs or GFP genes, respectively. Sequence analysis and comparison were then carried out to determine the recombinant plasmid pET-28a-CTP with the correct sequence Tat -JDlys, pET-28a-CTP Ant -JDlys, pET-28a-CTP TP10 -JDlys and pET-28a-GFP-CTP Tat -JDlys. Transform the above-mentioned recombinant plasmid into the expression recipient bacteria-Escherichia coli BL21(DE3), screen positive clones for induced expression and identification of the protein, and obtain recombinant JDlys protein and chimeric lyase recombinant protein CTP Tat -JDlys, CTP Ant -JDlys, CTP TP10 -JDlys and GFP-CTP Tat -JDlys, th...

Embodiment 3

[0056] Embodiment 3 Recombinant lyase in vitro minimum inhibitory concentration (MIC) determination

[0057] The recombinant expressed protein obtained in Example 2 was subjected to an in vitro lysis test to check the lysis activity and lysis spectrum. The specific operation is as follows:

[0058] Staphylococcus strains of different origins were grown to early logarithmic phase (OD600 of 0.2 to 0.4). After washing three times with PBS, take 100 μl of bacterial suspension (diluted to 5×10 6 cfu / mL) was mixed with 100 μl of recombinant lyase at different final concentrations (10, 20, 40, 80, 160, 320 and 640 μg / mL), and the MIC of the lyase was determined in a 96-well microtiter plate. Plates were incubated at 37°C for 20 hours and read with a 96-well plate reader (OD 600 ).

[0059] The experimental results are shown in Table 2, Tat type chimeric recombinant lyase CTP Tat -JDlys showed a slightly reduced bactericidal effect on a small number of strains compared with the...

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Abstract

The invention provides a chimeric enzyme antibiotic for killing staphylococcus aureus and the preparation and application thereof. The chimeric enzyme antibiotic comprises recombinant protein CTP-JDlys which is formed by connecting cytoplasmic transduction peptide CTP and staphylococcus aureus phage lyase JDlys in series. The preparation method of the chimeric enzyme antibiotic comprises the following steps of: S1, designing a primer, and respectively using a lyase JDlys gene and a cytoplasmic transduction peptide CTP gene as templates to amplify a JDlys gene fragment and a CTP gene fragment;S2, constructing a recombinant plasmid from the amplified JDlys gene, and inserting the amplified CTP gene into the 5'end of the JDlys gene in the recombinant plasmid to construct the recombinant plasmid containing a target gene fragment CTP-JDlys; S3, expressing and purifying the recombinant plasmid containing the target gene fragment CTP-JDlys to obtain the chimeric enzyme antibiotic protein CTP-JDlys. The CTP-JDlys protein can enter skin keratinocytes to effectively treat mouse skin abscess caused by drug-resistant staphylococcus aureus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation of a novel enzyme antibiotic in the field of biotechnology and its application in the treatment of bacterial infection in epidermal keratinocytes, and specifically relates to a class of bacteria that can efficiently enter eukaryotic cells to kill drug-resistant Staphylococcus aureus The biosynthesis of a novel chimeric enzyme antibiotic and the evaluation of its bactericidal effect on intracellular and skin abscesses; more specifically, it relates to a chimeric enzyme antibiotic that kills Staphylococcus aureus and its preparation and application. Background technique [0002] Skin and skin structure infections (SSSIs) are inflammatory diseases caused by various suppurative pathogenic bacteria invading the epidermis, dermis and subcutaneous tissue. There are many pathogens that cause SSSIs, including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/62C12N15/70C12Q1/02A61K49/00A61K38/51A61P31/04
CPCA61K38/00A61P31/04C07K2319/10C12N9/88C12N15/70G01N33/5008
Inventor 孙建和王兆飞严亚贤孔里程
Owner SHANGHAI JIAO TONG UNIV
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