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Recombinant engineering bacterium capable of efficiently expressing plectasin and application of recombinant engineering bacterium

A technology of recombining engineering bacteria and mycelia, which is applied in the field of genetic engineering, can solve the problems of unsatisfactory expression, high cost, and high impurity content, and achieve good application prospects, increased expression, and significant effects

Pending Publication Date: 2022-07-29
OCEAN UNIV OF CHINA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction and separation process of antimicrobial peptides obtained directly from natural resources is complicated and costly, and the yield of antimicrobial peptide products is low and the content of impurities is high. Therefore, it is very important to find an efficient method for expressing antimicrobial peptides
[0003] At present, the expression of plectasin in Pichia pastoris GS115 has been successfully achieved, but its expression level has not reached the ideal level, so it is necessary to develop a method that can efficiently express plectasin

Method used

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  • Recombinant engineering bacterium capable of efficiently expressing plectasin and application of recombinant engineering bacterium
  • Recombinant engineering bacterium capable of efficiently expressing plectasin and application of recombinant engineering bacterium
  • Recombinant engineering bacterium capable of efficiently expressing plectasin and application of recombinant engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Design of a T2A peptide-based tetraploid pleomycin tetramer

[0041] Based on the obtained gene sequence of pectin and the gene sequence of T2A peptide (GenBank: UER86417.1). A 6×His tag was added to the N-terminus of each pectin for purification. The T2A peptide sequence was added to the C-terminus of plucuronidin to connect with the next set of gene expression cassettes, and in this way, a quadruple concatemer of plucuronidin was constructed (the construction diagram is shown in Fig. figure 1 , 2 shown). The base sequence was optimized according to the codon usage preference of Pichia pastoris, and the whole gene was synthesized by Beijing Huada Gene Technology Co., Ltd. The nucleotide sequence of the optimized quadruple concatemer is shown in SEQ ID NO.5, wherein, the gene of pectin is shown in SEQ ID NO.1, encoding 40 amino acids, as shown in SEQ ID NO.2 .

[0042] SEQ ID NO. 5:

[0043]5’-ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTC...

Embodiment 2

[0044] Example 2 Cloning of plectomycin gene

[0045] Using the above-synthesized tetraploid gene fragment of phleomycin as a template, primers for seamless ligation were designed upstream and downstream of the phlegomycin gene, and PCR amplification of the phlegomycin gene fragment was performed.

[0046] The sequences of primers are as follows:

[0047] Upstream primer: 5'-GCTACGTAGAATTCGGTTTCGGTTGTAAC-3', as shown in SEQ ID NO.6;

[0048] Downstream primer: 5'-CGCTTAATGATGATGATGATGATGGTAGCACTTACAGACG-3', as shown in SEQ ID NO.7.

[0049] The primers for seamless ligation were designed upstream and downstream, and PCR was performed to amplify the gene fragment of the pPIC9k cloning vector.

[0050] The sequences of primers are as follows:

[0051] Upstream primer: 5'-CATCATCATTAAGCGGCCGCGAATTA-3', as shown in SEQ ID NO.8;

[0052] Downstream primer: 5'-GAATTCTACGTAAGCTTCAGCCTCTC-3' as shown in SEQ ID NO.9.

[0053] The PCR reaction system was: 2×PCR Buffer 25 μl, dNTP 1...

Embodiment 3

[0056] Example 3 Construction of the expression vector of the pecomycin gene

[0057] The pleomycin quadruple concatemer gene fragment was connected with the pPIC9k cloning vector using seamless cloning technology, and the ligated product was transferred into E.coli DH5α competent cells were plated on solid plates of (LB) medium containing 50 μg / mL kanamycin. After culturing in a 37°C incubator for 15 hours, pick a single clone into LB liquid medium containing 50 μg / mL kanamycin, and cultivate overnight in a 37°C shaker at a speed of 220 rpm. After positive verification, sequencing was performed, and the resulting expression vector was named as pPIC9k-Plectasin.

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Abstract

The invention discloses a recombinant engineering bacterium for efficiently expressing plectasin, the genome of the recombinant engineering bacterium contains a plectasin quadruple concatemer, the plectasin quadruple concatemer is formed by connecting four gene segments for encoding plectasin in series through gene segments for encoding T2A peptide, and the nucleotide sequence of the plectasin quadruple concatemer is shown as SEQ ID NO.5. The construction method comprises the following steps: adding a 6 * His tag at the N end of plectasin for purification, adding a T2A peptide sequence at the C end of plectasin for connection with a next group of gene expression cassettes, and constructing a quadruple concatemer of plectasin in this way; and transforming the recombinant expression vector into a host competent cell to obtain the recombinant engineering bacterium. The recombinant engineering bacterium capable of efficiently expressing plectasin is applied to preparation of plectasin, inhibition of staphylococcus aureus and preparation of a preparation for inhibiting staphylococcus aureus. The recombinant engineering bacterium can efficiently express plectasin and has a good application prospect.

Description

technical field [0001] The invention relates to a recombinant engineering bacterium with high expression of lectin and its application, and belongs to the technical field of genetic engineering. Background technique [0002] Antimicrobial peptides are an important part of the body's natural immune system and are often a key line of defense against pathogens. Antimicrobial peptides are considered to be the best substitutes for traditional antibiotics due to their high antibacterial activity, safety and unique mechanism of action. . The extraction and separation process of antimicrobial peptides obtained directly from natural resources is complicated, the cost is high, and the yield of antimicrobial peptide products is low and the content of impurities is high. Therefore, it is crucial to seek an efficient method for expressing antimicrobial peptides. [0003] At present, the expression of pecomycin in Pichia pastoris GS115 has been successfully achieved, but its expression ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/31C12N15/62C12R1/84
CPCC07K14/37C12N15/815C07K2319/21
Inventor 毛相朝姜宏司先懂梁星星董格菲任泽林陈鹏
Owner OCEAN UNIV OF CHINA
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