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CN-121991993-A - Method for improving expression quantity of bacterial strain lysostaphin

CN121991993ACN 121991993 ACN121991993 ACN 121991993ACN-121991993-A

Abstract

The invention provides a method for improving the expression quantity of bacterial strain lysostaphin, which belongs to the field of biotechnology, and specifically comprises the steps of transferring plasmid pET-28a-lacUV5 (49) -T7RNAP and plasmid pET-22b-lys containing lys coding lysostaphin gene into expression bacterial strain EcN delta P to improve the expression quantity of bacterial strain lysostaphin, wherein the nucleotide sequence of plasmid pET-28a-lacUV5 (49) -T7RNAP is SEQ ID NO.1, the amino acid sequence of lysostaphin is SEQ ID NO.2, and expression bacterial strain EcN delta P is ESCHERICHIA COLI NISSLE1917 of knock-out plasmids pMUT1 and pMUT 2.

Inventors

  • TONG HUAIZHOU
  • XIANG DONGLIANG
  • XU ZHENSHANG
  • Tong Huaiyu
  • XU NAN

Assignees

  • 江苏雪豹日化有限公司

Dates

Publication Date
20260508
Application Date
20260326

Claims (10)

  1. 1. A method for increasing expression level of lysostaphin a strain, which is characterized in that the method comprises the steps of transferring plasmid pET28a-lacUV5 (49) -T7RNAP and plasmid pET22b-lys containing lys encoding lysostaphin gene into expression strain EcN Δp to increase expression level of lysostaphin the strain, and the method comprises the following steps: a, connecting coding gene lys of lysostaphin to Nde I and Xho I sites of plasmid pET-22b (+) to obtain plasmid pET22b-lys, converting EcN delta P from plasmid pET28a-lacUV5 (49) -T7RNAP and plasmid pET22b-lys, and screening to obtain recombinant bacteria EcN delta P/pET28a-lacUV5 (49) -T7 RNAP/pET22b-lys; b fermenting recombinant bacteria EcN delta P/pET28a-lacUV5 (49) -T7RNAP/pET22b-lys to prepare lysostaphin; The nucleotide sequence of the plasmid pET28a-lacUV5 (49) -T7RNAP is SEQ ID NO.1; the amino acid sequence of the lysostaphin is SEQ ID NO.2; The expression strain EcN Δp is ESCHERICHIA COLI NISSLE 1917 from which plasmids pMUT1 and pMUT2 were knocked out.
  2. 2. The method of claim 1, wherein plasmids pMUT1 and pMUT2 in ESCHERICHIA COLI NISSLE 1917 are knocked out using the pren 112 plasmid to yield expression strain EcN Δp.
  3. 3. The method of claim 1, wherein the nucleotide sequence encoding a lysostaphin gene is SEQ ID No.3.
  4. 4. A mutant of the lacUV5 promoter, characterized in that the nucleotide sequence of said mutant is SEQ ID No.4, designated lacUV5 (49).
  5. 5. An E.coli Nissle 1917 expression system is characterized by comprising an expression strain EcN delta P, a plasmid pET28a-lacUV5 (49) -T7RNAP and an expression vector pET-22b (+); The nucleotide sequence of the plasmid pET28a-lacUV5 (49) -T7RNAP is SEQ ID NO.1; The expression strain EcN Δp is ESCHERICHIA COLI NISSLE 1917 from which plasmids pMUT1 and pMUT2 were knocked out.
  6. 6. The method for constructing an expression system of escherichia coli Nissle 1917 according to claim 5, comprising the steps of: The plasmid pET28a-lacUV5 (49) -T7RNAP and the expression vector pET-22b (+) were transformed into the expression strain EcN DeltaP.
  7. 7. The method according to claim 6, wherein plasmids pMUT1 and pMUT2 in ESCHERICHIA COLI NISSLE 1917 are knocked out using pRE112 plasmid to obtain expression strain EcN ΔP.
  8. 8. Use of the escherichia coli Nissle 1917 expression system of claim 5 for preparing an organic acid, a protein, an amino acid or a polysaccharide.
  9. 9. The high-yield lysostaphin strain is characterized by comprising an expression strain EcN delta P, a plasmid pET28a-lacUV5 (49) -T7RNAP and an expression vector pET-22b-lys; The nucleotide sequence of the plasmid pET28a-lacUV5 (49) -T7RNAP is SEQ ID NO.1; the amino acid sequence of the lysostaphin is SEQ ID NO.2; The expression strain EcN Δp is ESCHERICHIA COLI NISSLE 1917 from which plasmids pMUT1 and pMUT2 were knocked out.
  10. 10. The method for constructing a high-yield strain of lysostaphin claim 9, comprising the steps of: connecting coding gene lys of lysostaphin to Nde I and Xho I sites of plasmid pET-22b (+) to obtain plasmid pET22b-lys, converting EcN delta P from plasmid pET28a-lacUV5 (49) -T7RNAP and plasmid pET22b-lys, and screening to obtain recombinant bacteria EcN delta P/pET28a-lacUV5 (49) -T7 RNAP/pET22b-lys; preferably, plasmids pMUT1 and pMUT2 in ESCHERICHIA COLI NISSLE 1917 are knocked out by pRE112 plasmid to obtain an expression strain EcN delta P; preferably, the coding gene sequence of the lysostaphin is SEQ ID NO.3; Preferably, the lysostaphin gene is obtained by amplifying the lysostaphin gene sequence using the primer lys-F as shown in SEQ ID NO.23 and lys-R as shown in SEQ ID NO. 24.

Description

Method for improving expression quantity of bacterial strain lysostaphin Technical Field The invention belongs to the technical field of biology, and particularly relates to a method for improving expression quantity of bacterial strain lysostaphin. Background Coli expression systems are currently the most commonly used recombinant protein expression systems in genetic engineering, and because of their clear genetic background and simple genetic manipulation techniques, escherichia coli becomes one of the most common model organisms in genetic engineering bacteria and molecular biology research. Conventional E.coli expression systems need to include expression vectors and expression strains. The expression vector should have, in addition to the properties possessed by the cloning vector, the expression elements, i.e.the DNA sequences necessary for transcription and translation. Expression elements required for expression of a particular function of the expression vector include an origin of replication, a multiple cloning site, a transcription termination signal, a fusion tag, a resistance gene, and a promoter. The promoter on the expression vector is used as the recognition sequence of transcription initiation of the heterologous expression gene, is one of the key gene elements of the heterologous gene, can efficiently start the transcription process and directly affects the expression level of the target gene. The constitutive promoters can be classified into constitutive promoters and inducible promoters according to their regulatory characteristics, and the constitutive promoters can be continuously expressed without a specific inducer to induce their expression functions, but have the greatest disadvantage of failing to precisely regulate the reaction. Instead, the inducible promoter can precisely change the expression intensity of the target gene by stimulation of external factors such as changes in ambient temperature, changes in pH, addition of specific inducers, and changes in concentration. In the commercial plasmid pET series expression vectors, there is a promoter of RNA polymerase from T7 phage-T7 promoter. The T7 promoter, as a typical representation in inducible promoters, is capable of being efficiently recognized by a characteristic T7RNA polymerase (T7 RNAP) and initiates transcription at levels 3-5 times that of other promoters. In particular, the activity of T7RNAP is completely dependent on the presence of T7 promoter, and T7RNAP does not initiate transcription without T7 promoter, which ensures high selectivity and controllability of transcription process, avoiding nonspecific transcription and potential gene expression interference. The T7 promoter and T7RNAP serve as the main functional elements in the T7 expression system and tightly control the transcription process under the influence of other regulatory elements. The selection of the expression strain is critical to the expression of the heterologous protein, and the escherichia coli is used as a popular engineering strain at present, so that the characteristics of high expression level, high-purity protein and the like occupy a place in the synthesis biology. Strain ESCHERICHIA COLINISSLE 1917 (EcN), which belongs to a few probiotic model organisms among gram-negative bacteria, is traditionally used for preventing and treating various inflammatory diseases and is circulated as a commercial medicament of a probiotic preparation. Probiotics have great potential to be used as synthetic biology in metabolic engineering due to their high biosafety, non-toxicity, biocompatibility and good acceptability. The efficient heterologous expression capability of the T7 system allows researchers to deepen the exploration of the application level of the system and apply the system to various host strains, so that the efficient expression of heterologous genes is realized. In the prior art, the T7RNA polymerase gene is mainly integrated on the genome for expression, so that the operation is complicated, the period is too long, and the screening workload of the genome insertion site is too large. And integrated into the genome, is inconvenient for the micro-regulation of the expression of T7RNA polymerase. The research and development of the escherichia coli expression system which is simple to operate and has high-efficiency heterologous expression capability has important functions. The lysostaphin has high specificity and safety, and shows important value in the fields of drug-resistant bacteria prevention and control, food preservation and bioengineering. Lysostaphin can specifically recognize and hydrolyze glycine pentapeptide bridging structure (existing in peptidoglycan layer) in staphylococcal cell wall, resulting in cell wall rupture and bacterial death. In the field of food industry, the cell wall of staphylococcus (such as staphylococcus aureus) is directly destroyed, the growth and reproduction of the staphylococcus (such as staphyloc