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Wide spectrum bacteriophage chimeric lytic enzyme capable of resisting staphylococcus, preparation method and appliance thereof

A staphylococcus and lyase technology, which is applied in the field of broad-spectrum phage chimeric lyase and its preparation, can solve problems such as hindering the application of antimicrobial peptides, high cost of chemical synthesis, and disrupting balance, and achieves safety, no toxic side effects, and high prevention and control. effect of effect

Active Publication Date: 2016-01-13
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the large-scale application of antimicrobial peptides is limited by the following two points: First, the correct paired disulfide bonds can be obtained by using chemical synthesis technology, but the high cost of chemical synthesis hinders the production of antimicrobial peptide molecules, so it is difficult to produce on a large scale The second is that antimicrobial peptides can non-selectively attack various microorganisms such as viruses, fungi, Gram-negative bacteria and Gram-positive bacteria, and destroy the balance of normal flora in the body while killing pathogenic bacteria. Produces certain cytotoxicity to mammals, thus hindering the further application of antimicrobial peptides
At present, the combination of phage lytic enzyme and antimicrobial peptide gene for genetic modification has not been reported for the prevention and treatment of human and animal infections caused by various staphylococci.

Method used

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  • Wide spectrum bacteriophage chimeric lytic enzyme capable of resisting staphylococcus, preparation method and appliance thereof
  • Wide spectrum bacteriophage chimeric lytic enzyme capable of resisting staphylococcus, preparation method and appliance thereof
  • Wide spectrum bacteriophage chimeric lytic enzyme capable of resisting staphylococcus, preparation method and appliance thereof

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Experimental program
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Effect test

Embodiment 1

[0044] Cloning of embodiment 1 recombinant chimeric lyase AP-LyS1 gene, construction of expression vector

[0045] (a) The chimeric lyase AP-LyS1 gene was designed according to the structural characteristics of the staphylococcus cell wall and cell membrane and the lyase gene sequence of the staphylococcus virulent phage JS25 isolated in our laboratory, and its nucleotide sequence is shown in SeqID NO.1.

[0046] (b) The chimeric lyase AP-LyS1 gene was handed over to Nanjing GenScript Biosynthetic Gene and subcloned to obtain the recombinant expression plasmid pET32a-AP-LyS1, and the recombinant expression plasmid was introduced into and transformed Escherichia coli TOP10 competent cells (purchased from Tiangen Biochemical Technology Co., Ltd., the specific operation steps are carried out according to the product instructions), and then spread the transformed Escherichia coli on an LB plate containing ampicillin (50 μg / ml), cultivate overnight at 37 ° C, and pick positive clone...

Embodiment 2

[0048] Example 2 Induced expression and purification of chimeric lyase AP-LyS1 protein

[0049] Inoculate the recombinant strain BL21 (pET32a-AP-LyS1) into 4ml LB liquid medium containing ampicillin (50μg / mL), culture overnight at 37°C with shaking; the next day, transfer to 100mL LB liquid medium at a ratio of 1:100 , cultured with shaking at 37°C until OD 600 When the value is 0.3, add IPTG to the final concentration of 0.5mmol / L, and induce for 20h at 20°C; then collect the bacteria, and ultrasonically disrupt the cells (ultrasonic program: work for 3s, pause for 3s, 50% power, and the total ultrasonic time is 5min); Centrifuge at 10,000 rpm for 10 min to collect the supernatant; then purify the supernatant with a His affinity chromatography nickel column (purchased from GE Healthcare, Sweden) (the specific steps are carried out according to the kit instructions), and the obtained protein is named as cleavage Enzyme AP-LyS1;

[0050] Analysis results such as figure 2 As...

Embodiment 3

[0052] The analysis of the bactericidal spectrum of embodiment 3 chimeric lyase AP-LyS1

[0053] The following bacteria: Staphylococcus aureus ATCC25923, CMCC26001, JYG1, JYG2, YZ-34, YZ-42, YZ-49, YZ-56, XG-9, XB-2, TQ1, TQ2, SF-37, SF- 46. ​​SF-54, SF-56, 56A, MP0070, S4, S6; Staphylococcus epidermidis 975, 987, 999, S09099; Staphylococcus hemolyticus (946); Staphylococcus chromogenes (S10165); Staphylococcus amberi (S07117) Streptococcus agalactiae (BAA61); Salmonella (S10177); Klebsiella pneumoniae (K10170); Enterococcus (29); Escherichia coli (E10170);

[0054] Insert into TSB medium respectively, cultivate overnight at 37°C, and then take 0.1ml of overnight culture (bacteria content 10 9 cfu / ml) was added to the TSB plate,

[0055] Then take 0.01mL of the purified lyase AP-LyS1 product obtained in Example 2 (concentration 100ug / ml) and drop it in the center of the TSB plate, spread the bacterial solution evenly, incubate at 37°C for 1 hour, and let it dry naturally Af...

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Abstract

The invention discloses a wide spectrum bacteriophage chimeric lytic enzyme capable of resisting staphylococcus, wherein a nucleotide sequence wide spectrum bacteriophage chimeric lytic enzyme capable of resisting the staphylococcus as shown in sequence identifier number 1 (Seq ID NO.1), an amino acid sequence as shown in Seq ID NO.2. The wide spectrum bacteriophage chimeric lytic enzyme capable of resisting the staphylococcus is used to prevent various staphylococcus, can be separately used or matched to use, is used to prevent various pollutions and infections caused by the staphylococcus, has extremely high prevention effect to cow mastitis infection caused by the staphylococcus in dairy industry, and is safe without side effects.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an anti-staphylococcus broad-spectrum phage chimeric lyase and its preparation method and application. Background technique [0002] Staphylococcus is widely distributed in the air, drinking water, ground, surface and feed. The skin, mucous membranes, intestinal tract, respiratory tract and mammary glands of humans, livestock and poultry are also parasitic. Pathogenic staphylococci cause various suppurative infections, sepsis and sepsis, and can cause food poisoning when they contaminate food. According to different biochemical reactions and pigment production, there are more than 20 kinds of Staphylococcus, among which Staphylococcus aureus is an important zoonotic pathogen, which can cause wound infection, endocarditis, osteomyelitis, poisoning, etc. Shock syndrome and other diseases. Staphylococci other than Staphylococcus aureus can be called coagulase-negative staphyloe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/70A61K38/51A61P31/04A61P15/14
CPCA61K38/00C12N9/88
Inventor 孙利厂王冉魏瑞成
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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