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CN-121975832-A - Construction method of genetically engineered bacterium X33-H-CZS-4 for expressing fusion peptide H-CZS-4

CN121975832ACN 121975832 ACN121975832 ACN 121975832ACN-121975832-A

Abstract

The invention discloses a construction method of gene engineering bacteria X33-H-CZS-4 for expressing fusion peptide H-CZS-4, which comprises the steps of firstly preparing a fragment with HSAD-D3 label, secondly preparing a fragment containing complete CZS-4 coding sequence, assembling the fragment and the fragment in the first step in competent cells of escherichia coli Top10 to construct expression plasmid pPICZalphaA-HSAD2-D3-CZS-4, thirdly amplifying and culturing Pichia pastoris X33 to prepare competent cells, fourthly cutting and linearizing the obtained expression plasmid pPICZalphaA-HSAD-D3-CZS-4 and converting the obtained expression plasmid with the competent cells of Pichia pastoris X33 to obtain gene engineering bacteria X33-H-CZS-4 for expressing fusion peptide H-CZS-4, and successfully constructing gene engineering bacteria X33-H-CZS-4 for expressing fusion peptide H-CZS-4 by integrating the peptide H-CZS-4 with HSAD-D3 label into the competent cells of Pichia pastoris X33.

Inventors

  • ZHAO CHEN
  • SONG JIA
  • ZHANG JIAYI
  • SUN BO
  • LIU KUANBO

Assignees

  • 国家粮食和物资储备局科学研究院

Dates

Publication Date
20260505
Application Date
20260108
Priority Date
20251208

Claims (6)

  1. 1. The construction method of the gene engineering bacteria X33-H-CZS-4 for expressing fusion peptide H-CZS-4 is characterized by comprising the following steps of: step one, designing a pair of primers to carry out PCR amplification by taking pPICZalphaA-HSAD2-D3-CLP1 opt Pichia plasmid containing HSAD-D3 tag as a template to obtain a fragment with HSAD2-D3 tag; Designing another pair of primers, obtaining a fragment containing a complete CZS-4 coding sequence by an overlap extension PCR amplification method, and assembling the fragment and the fragment in the first step in competent cells of escherichia coli Top10 to construct an expression plasmid; Amplifying and culturing engineering bacteria Pichia pastoris X33 to prepare Pichia pastoris competent cells, and sub-packaging and refrigerating for later use; And fourthly, carrying out enzyme digestion linearization on the obtained expression plasmid pPICZalphaA-HSAD2-D3-CZS-4 and transforming with competent cells of Pichia pastoris X33 to obtain the genetically engineered bacterium X33-H-CZS-4.
  2. 2. The method for constructing genetically engineered bacterium X33-H-CZS-4 expressing fusion peptide of claim 1, wherein pPICZalphaA-HSAD2-D3-CLP1 opt Pichia is obtained by genetic synthesis and stored in a refrigerator at-20deg.C, and the nucleotide sequence is shown in SEQ ID NO. 2.
  3. 3. The method for constructing H-CZS-4 gene engineering bacteria X33-H-CZS-4, wherein the Primer pair in the first step is Primer1 and Primer2 with nucleotide sequences shown as SED ID NO.3 and SED ID NO.4 respectively, and the Primer pair is used for amplifying a DNA fragment with the length of 1301bp on pPICZalphaA-HSAD2-D3-CLP1 opt Pichia, wherein the fragment comprises HSAD-D3 tag.
  4. 4. The method for constructing H-CZS-4 gene engineering bacteria X33-H-CZS-4, according to claim 1, wherein the Primer pair in the second step is Primer3 and Primer4 nucleotide sequences respectively shown as SED ID No.5 and SED ID No.6, and a DNA fragment with the length of 102bp is amplified by an overlap extension PCR method, wherein the fragment contains a complete CZS-4 coding sequence.
  5. 5. The method for constructing genetically engineered bacterium X33-H-CZS-4 expressing fusion peptide of claim 1, wherein the E.coli vector in the second step is pPICZalphaA comprising AOX1 promoter, a purification tag of 6XHis, and a resistance gene Bleo R .
  6. 6. The method for constructing H-CZS-4 gene engineering bacteria X33-H-CZS-4, which is characterized in that the mixed transformation method in the fourth step is one of an electrochemical transformation method and a chemical transformation method, and after transformation, the PCR verification is carried out on the transformant by using a Primer pair 5 and a Primer6, so as to obtain the H-CZS-4 fusion peptide expression bacterial strain X33-H-CZS-4, wherein the nucleotide sequences of the Primer pair are shown as SED ID NO.7 and SED ID NO. 8.

Description

Construction method of genetically engineered bacterium X33-H-CZS-4 for expressing fusion peptide H-CZS-4 Technical Field The invention relates to the technical field of molecular biology, in particular to a construction method of fusion peptide H-CZS-4 genetically engineered bacteria. Background Unlike traditional antibiotics, which mainly act on specific metabolic targets (such as cell wall synthesis or protein translation process), most antibacterial peptides realize rapid sterilization by physically destroying the integrity of microbial cell membranes, and the unique action mechanism remarkably reduces the risk of microbial drug resistance, so that the antibacterial peptides are considered as one of the possible schemes for resisting increasingly severe antibiotic drug resistance, and have become the key direction for the development of new-generation antibacterial drugs. The antibacterial peptide CZS-4 extracted from the rana chensinensis has 23 amino acids and three conserved amino residues, namely Glycine (Glycine), leucine (Leucine) and Lysine (Lysine), wherein the Glycine and the leucine are hydrophobic amino acids, the Lysine is a positively charged basic amino acid, the synergistic effect of the three amino acids can regulate the amphiphilicity balance and net positive charge number of the CZS-4, the molecular weight of the CZS-4 is 24229.89Da, the CZS-4 is positively charged at physiological pH value, and isoelectric points higher than 7 can be better combined with cell membranes of negatively charged bacteria, so that the bacterial structure is destroyed. At present, in the method for obtaining the antibacterial peptide CZS-4, the steps of directly separating from organisms are complicated, the separation rate is low, and the chemical synthesis faces the bottleneck of high cost and limited efficiency, so the invention provides a construction method for expressing fusion peptide H-CZS-4 genetic engineering bacteria X33-H-CZS-4 to solve the problems in the prior art. Disclosure of Invention Aiming at the problems, the invention aims to provide a construction method of a genetically engineered bacterium X33-H-CZS-4 for expressing fusion peptide H-CZS-4, which is implemented by integrating HSAD-D3 tag and a coding sequence of the antibacterial peptide CZS-4 into a pPICZalphaA expression vector and transforming the vector with pichia pastoris X33 competent cells. The invention aims at realizing the aim by adopting the following technical scheme that the construction method for expressing fusion peptide H-CZS-4 (HSAD-D3-CZS-4 is short for short) genetic engineering bacteria X33-H-CZS-4 is realized, and the amino acid sequence of the antibacterial peptide CZS-4 is shown as SEQ ID NO.1, and comprises the following steps: step one, designing a pair of primers to carry out PCR amplification by taking pPICZalphaA-HSAD2-D3-CLP1 opt Pichia plasmid containing HSAD-D3 tag as a template to obtain a fragment with HSAD2-D3 tag; designing another pair of primers, obtaining a fragment containing a complete CZS-4 coding sequence by an overlap extension PCR amplification method, and assembling the fragment and the fragment in the step one in competent cells of escherichia coli Top10 to construct an expression plasmid; Step three, performing amplification culture on pichia pastoris X33 to prepare pichia pastoris competent cells, and sub-packaging and refrigerating for standby; And fourthly, carrying out enzyme digestion linearization on the obtained expression plasmid pPICZalphaA-HSAD2-D3-CZS-4 and transforming with competent cells of Pichia pastoris X33 to obtain the gene engineering bacterium 33-pPICZalphaA-HSAD2-D3-CZS-4 (X33-H-CZS-4 for short) for expressing the fusion peptide. The further improvement is that pPICZalphaA-HSAD2-D3-CLP1 opt Pichia is obtained by gene synthesis and stored in a refrigerator at-20 ℃ and the nucleotide sequence is shown as SEQ ID NO. 2. The further improvement is that the Primer pair in the first step is Primer1 and Primer2 nucleotide sequences respectively shown as SED ID NO.3 and SED ID NO.4 and is used for amplifying a DNA fragment (with HSAD2-D3 tag fragment) with the length of 1301bp on pPICZalphaA-HSAD2-D3-CLP1 opt Pichia. The further improvement is that the Primer pair in the second step is Primer3 and Primer4 nucleotide sequences respectively shown as SED ID No.5 and SED ID No.6, and DNA fragments (with complete CZS-4 coding sequences) with the length of 102bp are amplified by an overlap extension PCR method. The further improvement is that the escherichia coli vector in the second step is pPICZalphaA containing AOX1 promoter, a purification tag of 6xHis, a resistance gene Bleo R and other elements. The method is further improved in that the conversion method in the step four is one of an electrochemical conversion method and a chemical conversion method, after conversion, the Primer5 and the Primer6 are used for carrying out PCR verification on the transformant, so that the ge