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Construction method and application of attenuated Listeria monocytogenes

A Listeria and monocyte technology, applied in the field of genetic engineering, can solve the problems of easy transfer and recurrence, long immunization cycle, poor immunization effect, etc., to eliminate plasmid loss, stable attenuated strains, and good immunogenicity Effect

Pending Publication Date: 2020-06-12
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The traditional methods of treating cancer are surgical resection, radiotherapy, chemotherapy and therapeutic vaccines. Surgical resection, radiotherapy and chemotherapy are prone to disadvantages such as metastasis and recurrence. In contrast, vaccine therapy has the advantage of less damage to the body and low toxicity. However, the vaccines used in China are mainly traditional inactivated vaccines and subunit vaccines, which have disadvantages such as long immunization cycle and poor immunization effect.

Method used

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  • Construction method and application of attenuated Listeria monocytogenes
  • Construction method and application of attenuated Listeria monocytogenes
  • Construction method and application of attenuated Listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The construction of embodiment 1 attenuated vaccine vector

[0051] 1. Construction of recombinant plasmids

[0052] From Listeria monocytogenes wild strain EGD-e bacterial strain (ATCC standard bacterial strain), directly clone required fragment, use Vector NTI Explorer to design amplification primer pSL279-Sal I-F and pSL279-BamH I-R, full-length 819bp (as SEQ ID NO .1), related primers are shown in Table 1:

[0053] Table 1 Primers required for gene amplification and verification of EGD-e hly

[0054]

[0055] Note: pSL279-Sal I-F is the upstream primer, pSL279-BamH I-R is the downstream primer, homoarms front is the primer used for verification at a distance of 63bp from the homology arm on the Listeria genome, M13-F is the upstream verification primer on the pKSV7 plasmid, M13 -R is the downstream verification primer on the pKSV7 plasmid, pSL282-F is the upstream primer for constructing the spike, and pSL282-R is the downstream primer for constructing the spik...

Embodiment 2

[0063] Example 2 Attenuated strain phenotype analysis and infection biology analysis

[0064] 1. Growth ability analysis

[0065] Pick a single colony in 5ml of BHI liquid medium, place it in a shaker at 37°C for overnight culture, take 1ml of the bacterial solution to adjust the OD to 0.2, then dilute it 100 times with fresh BHI medium, take 200μl in a 96-well enzyme plate , 3 parallels for each bacterium, measure the optical density value (OD value) at 600nm with a microplate reader, place it in a constant temperature incubator at 37°C, measure once every 1h, and measure continuously for 12h. Depend on image 3 As shown, compared with the wild strain EGD-e, the growth ability of Lemo-C07 in BHI medium was not affected.

[0066] 2. LLO and its mutant LLO N478AV479A Protein expression assay

[0067] Pick a single colony in 5ml BHI liquid medium, place it on a shaker at 37°C for overnight culture, take 1ml of the bacterial liquid and transfer it to 100ml BHI liquid medium, ...

Embodiment 3

[0078] Immune effect evaluation of embodiment 3 attenuated strain

[0079] 1. Determination of transcriptional levels of immune factors in mouse macrophages

[0080] Plate Raw264.7 cells in a 6-well cell culture plate, culture overnight, infect the cells with Listeria at MOI=10:1, use PBS as a blank control, incubate at 37°C for 30min, and wash with 10mM PBS (pH 7.4) 2-3 times, add DMEM containing 50 μg / ml gentamicin to continue culturing, wash 2-3 times with 10 mM PBS (pH 7.4) after 30 min, add DMEM containing 10% FBS and 5 μg / ml gentamicin to continue Cultivate for 3h and 6h, digest the cells with 0.25% trypsin at each time point, centrifuge, take the cells, extract cellular RNA with a total cellular RNA extraction kit, and reverse into cDNA with a reverse transcription kit, RT-PCR Detect relevant inflammatory factors, relevant primers are shown in Table 2:

[0081] Table 2 Primers required for qPCR

[0082]

[0083] Note: F stands for upstream primer, R stands for dow...

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Abstract

The invention relates to the field of gene engineering, and aims to provide a construction method and application of attenuated Listeria monocytogenes. A Listeria monocytogenes wild strain EGD-e is used as a construction parent, and asparagine at the 478th site and valine at the 479th site of an hly gene are respectively mutated into alanine without containing plasmids; the finally obtained attenuated strain is named as Lemo-C07, the preservation number of the attenuated strain is CGMCC 18647, and the preservation institution is China General Microbiological Culture Collection Center. The attenuated strain of Listeria monocytogenes can be used as a live vaccine treatment carrier or an immunologic adjuvant; after amino acid site-specific mutagenesis of the key sites N478 and V479 of the Listeria monocytogenes virulence gene hly, the toxicity of the Listeria monocytogenes is greatly reduced (reaching a safety level), but very good immunogenicity is still retained. As direct mutation is carried out on the Listeria monocytogenes genome, the Listeria monocytogenes does not contain resistance plasmids, the biosafety is met, and the growth of the Listeria monocytogenes is not influenced by the loss of the plasmids.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a Listeria monocytogenes, specifically an attenuated Listeria monocytogenes, which can be used to deliver and express foreign antigens, and can be used as a live vaccine carrier . Background technique [0002] Listeria monocytogenes is a Gram-positive facultative anaerobic bacterium that can escape from the host cell phagosome into the host cell cytoplasm with the assistance of hemolysin O (LLO) and phospholipase C (PLC) , and recruit host actin aggregation through the virulence factor ActA to promote its migration and movement between host cells. Because Listeria has a unique intracellular parasitic life, it can be used as a live carrier vaccine to induce the secretion of a variety of important cytokines, such as IFN-γ, IL-4, IL-12 and IL-18. The generation of innate immunity can also be promoted by preferentially promoting antigen-specific CD4 + T cells and CD8 + T cells pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C07K16/00A61K39/02A61P31/04C12R1/01
CPCC07K14/195C12N15/74C07K16/00A61K39/0208A61P31/04A61K2039/522C12N1/205C12R2001/01C07K16/1296
Inventor 宋厚辉程昌勇俞慧飞孙静章先刘峰
Owner ZHEJIANG FORESTRY UNIVERSITY
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