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Construction method and application of novel attenuated listeria monocytogenes based on amino acid modification

A Listeria and amino acid technology, applied in the field of genetic engineering, can solve the problems of low antigen presentation efficiency, insufficient body safety, low immunogenicity, etc. Effect

Pending Publication Date: 2022-03-15
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the traditional attenuation strategy is mainly the knockout of virulence genes, plasmid complementation, etc., which have limitations such as insufficient safety for the body, low immunogenicity, and low antigen presentation efficiency.

Method used

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  • Construction method and application of novel attenuated listeria monocytogenes based on amino acid modification
  • Construction method and application of novel attenuated listeria monocytogenes based on amino acid modification
  • Construction method and application of novel attenuated listeria monocytogenes based on amino acid modification

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Experimental program
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Embodiment 1

[0041] The construction of embodiment 1 attenuated vaccine vector

[0042] 1. Construction of recombinant plasmids

[0043]Using the Listeria monocytogenes standard strain genome (GenBank sequence number is NC_003210.1) as a template, use Snapgene software to select amino acids 87-250 in the hly gene as the upstream homology arm, and select amino acids 256-422 in the hly gene amino acid as the downstream homology arm. Fragment A (501 bp, amplification primers are P2 and P3) and fragment B (499 bp, amplification primers are P4 and P5) respectively containing restriction sites BamH I and Pst I were amplified by PCR. On this basis, the target fragment of "A-B" (as shown in SEQ ID NO.1) was amplified by overlapping PCR technique (SOE-PCR). Using BamH I and Pst I as restriction sites, the recombinant plasmid pSL2424 was obtained by restriction enzyme digestion and enzyme ligation to the Listeria shuttle plasmid pKSV7, which was stored at -20°C after sequencing verification.

[0...

Embodiment 2

[0055] Example 2 Attenuated strain infection biological analysis

[0056] 1. Mouse organ proliferation test

[0057] Listeria monocytogenes was infected by intraperitoneal injection in 18-22g ICR female mice (infection amount was about 10 6 CFU), the mouse liver and spleen were isolated after 24 and 48 hours of infection, respectively, added 10mM PBS (pH 7.4) to fully grind, then diluted to an appropriate concentration, and placed on the plate and cultured at 37°C for 24 hours, then counted the bacterial colonies. The results were as follows: log 10 CFU presented. Such as figure 2 As shown, compared with WT, the colonization ability of LAPD5155 in mouse liver (Liver) and spleen (Spleen) was significantly decreased.

[0058] 2. Comparison of median lethal dose

[0059] Listeria monocytogenes was infected by intraperitoneal injection in 18-22g ICR female mice (infection amount was about 10 6 CFU), from the day of bacterial infection, observe the survival situation of the ...

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Abstract

The invention relates to the field of gene engineering, and aims to provide a construction method and application of novel attenuated listeria monocytogenes based on amino acid modification. The attenuated monocytosis listeria is obtained by taking a listeria monocytosis wild strain as a background and knocking out 251-255th amino acids in an hly gene. The invention provides an attenuated strain containing the attenuated listeria monocytogenes, and the attenuated strain is preserved in China General Microbiological Culture Collection Center (CGMCC). The attenuated strain of the listeria monocytogenes can be used as an exogenous antigen delivery live vector for immunotherapy or immunologic adjuvants of diseases such as tumors and the like. Due to the fact that knockout modification is carried out on the genome of the listeria monocytogenes, biomarkers such as resistant plasmids are not contained, biological safety is met, and in-vitro growth of the listeria monocytogenes is not affected.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a Listeria monocytogenes, specifically a novel Listeria monocytogenes, which can be used to deliver and express foreign antigens due to weakened virulence. Used as a live vaccine vector for immunotherapy. Background technique [0002] Listeria monocytogenes, referred to as Listeria monocytogenes, is a foodborne intracellular parasitic pathogen that is zoonotic. Listeria has a strong ability to survive and is widely distributed in various environments. It can continue to survive in many different environments. It is very easy to infect people with low immunity, causing bacterial sepsis, gastroenteritis, meningitis and abortion. Wait for listeriosis. [0003] In recent years, the research community has discovered through the study of Listeria that it can be used as a promising vaccine carrier to present tumor-associated antigens, and has been shown to be effective in human papillo...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/66C12N15/31A61K39/02A61P31/04C12R1/01
CPCC07K14/195C12N15/74A61K39/0208A61P31/04A61K2039/522A61K2039/523
Inventor 宋厚辉程昌勇孙静汪枫婷夏菁徐加利刘晨程茵蒋昕
Owner ZHEJIANG FORESTRY UNIVERSITY
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