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CN-122011215-A - Method for efficiently expressing and purifying lysozyme and application thereof

CN122011215ACN 122011215 ACN122011215 ACN 122011215ACN-122011215-A

Abstract

The invention provides a method for efficiently expressing and purifying lysozyme and application thereof, and relates to the technical field of bioengineering. The novel virulent phages vB_ SmaS _QH2 3 and vB_ SmaS _QH2 16 of the stenotrophomonas maltophilia (carbapenem antibiotics resistant) are subjected to genome sequencing, so that lysozyme QH3-15 and QH16-39 which are possibly effective to carbapenem gram-negative bacteria are found, codon optimization is carried out on the gene sequences of QH3-15 and QH16-39, the lysozyme QH3-15 and QH16-39 are cloned into a prokaryotic expression vector pET-28a and are overexpressed in an escherichia coli expression strain BL21 (DE 3) PlysS, and the lysozyme QH3-15 and QH16-39 are obtained through nickel column purification.

Inventors

  • CHENG PENG
  • LI ZIAN
  • ZHOU JIANWU
  • MOU XIAOMING
  • SUN JINGWEI
  • A XIANGREN

Assignees

  • 青海省人民医院

Dates

Publication Date
20260512
Application Date
20260327

Claims (10)

  1. 1. The high-efficiency expression purification lysozyme QH3-15 is characterized in that the lysozyme QH3-15 is obtained by adding a 6XHis tag into original QH3-15 protein, the amino acid sequence of the lysozyme QH3-15 is shown as SEQ ID NO.1, and the amino acid sequence of the original QH3-15 protein is shown as SEQ ID NO. 2.
  2. 2. The high-efficiency expression purification lysozyme QH16-39 is characterized in that the lysozyme QH16-39 is obtained by adding a 6XHis tag into original QH16-39 protein, the amino acid sequence of the lysozyme QH16-39 is shown as SEQ ID NO.3, and the amino acid sequence of the original QH16-39 protein is shown as SEQ ID NO. 4.
  3. 3. A fusion protein comprising the lysozyme QH3-15 or lysozyme QH16-39 of any one of claim 1 or claim 2, and an affinity tag linked to said lysozyme QH3-15 or lysozyme QH 16-39.
  4. 4. A polynucleotide encoding the lysozyme QH3-15 of claim 1, wherein the polynucleotide encoding the lysozyme QH3-15 has the sequence shown in SEQ ID No. 5; Or, the polynucleotide sequence for encoding the lysozyme QH16-39 as set forth in claim 2 is shown in SEQ ID NO. 6.
  5. 5. An expression vector comprising the polynucleotide of claim 4.
  6. 6. A host cell comprising the expression vector of claim 5.
  7. 7. A method for preparing the purified lysozyme QH3-15 or lysozyme QH16-39 with high expression according to any one of claims 1 or 2, comprising the steps of: s1, transforming the expression vector of claim 5 into a host cell; S2, inducing expression in a culture medium containing IPTG at 16 ℃; S3, collecting thalli, and obtaining supernatant containing target protein after pyrolysis; S4, purifying by affinity chromatography to obtain the lysozyme.
  8. 8. The method of claim 7, wherein the IPTG is at a concentration of 0.1mM to 0.5mM.
  9. 9. Use of a lysozyme according to any one of claims 1 to 2 in the manufacture of a medicament for inhibiting or killing multidrug-resistant gram-negative bacteria.
  10. 10. The use according to claim 9, wherein, the multi-drug resistant gram negative bacteria comprise any one or more of carbapenem-resistant enterobacteria, acinetobacter baumannii, pseudomonas aeruginosa or stenotrophomonas maltophilia.

Description

Method for efficiently expressing and purifying lysozyme and application thereof Technical Field The invention relates to the technical field of bioengineering, in particular to a method for efficiently expressing and purifying lysozyme and application thereof. Background Multiple drug resistant bacteria (MDRO) are bacteria that exhibit resistance to three or more classes of antimicrobial drugs simultaneously, and have become a significant threat to the world public health field. The common multi-drug resistant bacteria in hospital environment mainly comprise two major types of gram-positive bacteria and gram-negative bacteria. Wherein the gram positive bacteria are represented by methicillin-resistant staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), and the gram negative bacteria comprise bacteria of the family Enterobacteriaceae which produce ultra-broad-spectrum beta-lactamase (ESBLs), bacteria of the family Enterobacteriaceae which resist Carbapenem (CRE), multi-drug resistant pseudomonas aeruginosa (MDR-PA), multi-drug resistant Acinetobacter baumannii (MDR-AB) and the like. The clinical distribution of these resistant bacteria is characterized significantly. MRSA mainly causes skin soft tissue infection, pneumonia and blood flow infection, VRE is an important pathogen of hospital acquired infection and is easy to cause urinary tract infection and septicemia, ESBLs escherichia coli and klebsiella pneumoniae are commonly caused by respiratory tract infection and urinary system infection, CRE and CRAB mainly occur in an Intensive Care Unit (ICU) and often cause ventilator-related pneumonia and blood flow infection, and mortality rate is high. The global bacterial drug resistance situation is increasingly severe. According to a study result published by the international journal of authoritative medicine "lancet" in 9 of 2024, about 130 tens of thousands of people worldwide die from bacterial resistance in 2019. The treatment of multi-drug resistant bacterial infections presents a great challenge, primarily limited to the scarcity of available drugs and the rapid development of bacterial resistance. For CRE infections, particularly metallo-beta-lactamase (MBL) producing strains, conventional treatment regimens are extremely ineffective. Lysozyme has been discovered since 1922, and has undergone a recent century of research history. From the initial antibacterial enzymes to the present multifunctional biological molecules, the research of lysozyme gradually progresses from the initial discovery and basic characterization to the application research and industrialization. And finally, the application field of the lysozyme is continuously expanded, and the lysozyme is expanded from the original medical field to a plurality of fields such as food, daily chemicals, environment and the like. In a word, lysozyme is taken as a natural, safe and multifunctional biological enzyme, and has wide application prospect in various fields such as health, food, environment and the like. With the continuous deep research and the continuous innovation of the technology, the lysozyme has to play a more important role in the future technological and industrial development, and makes a greater contribution to human health and sustainable development. In view of this, the present invention has been proposed. Disclosure of Invention In order to solve the technical problems, the invention provides a method for efficiently expressing and purifying lysozyme and application thereof, which finds out lysozyme QH3-15 and QH16-39 which can be effective on carbapenem-resistant gram-negative bacteria by sequencing novel virulent phages vB_ SmaS _QH2 and vB_ SmaS _QH2 16 of stenotrophomonas maltophilia (carbapenem-resistant antibiotics) and performing genome sequencing, performs codon optimization on QH3-15 and QH16-39 gene sequences, clones into a prokaryotic expression vector pET-28a and is overexpressed in an escherichia coli expression strain BL21 (DE 3) PlysS, and obtains the lysozyme QH3-15 and QH16-39 by nickel column purification. In order to achieve the purpose of the invention, the following technical scheme is adopted: The invention provides a high-efficiency expression purified lysozyme QH3-15, which is characterized in that the lysozyme QH3-15 is obtained by adding a 6xHis tag into original QH3-15 protein, the amino acid sequence of the lysozyme QH3-15 is shown as SEQ ID NO.1, and the amino acid sequence of the original QH3-15 protein is shown as SEQ ID NO. 2. SEQ ID NO.1: MGSSHHHHHHSSGLVPRGSHMTNLPPTVKAKIDETIKAEGGAKVTNDPADRGGLTKYGVTQATWNAFREPSWPASVADATYEQAVEIYAKRYWLAPGFDRIATVSPPLAFEMFDWGVTSGPSRPVQALQRALNALNTQAKDYPDISADGVYGAMSHSALTAFRNRRAVEGLRYLLDMVQSLRRVFYIEISERDKTQERFQNGWQSRIK SEQ ID NO.2: MTNLPPTVKAKIDETIKAEGGAKVTNDPADRGGLTKYGVTQATWNAFREPSWPASVADATYEQAVEIYAKRYWLAPGFDRIATVSPPLAFEMFDWGVTSGPSRPVQALQRALNALNTQAKDYPDISADGVYGAMSHSALTAFRNRRAVEGLRYLLDMVQSLRRVFYIEISERDKTQERFQNGWQSRIK The invention also provides