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KRAS high-expression cancer vaccine based on recombinant attenuated Listeria monocytogenes and preparation method and application method thereof

A technology for Listeria and cancer vaccines, applied in the field of biomedicine, can solve the problem of low immunogenicity

Pending Publication Date: 2020-07-10
IMMORNA (HANGZHOU) BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The market needs a vaccine that makes up for the low immunogenicity of nucleic acid and protein peptide vaccines. The attenuated Listeria retains the ability of the original strain to grow intracellularly, completes the characteristics of antigen presentation, and at the same time has a higher Safe vaccine The present invention solves such problems

Method used

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  • KRAS high-expression cancer vaccine based on recombinant attenuated Listeria monocytogenes and preparation method and application method thereof
  • KRAS high-expression cancer vaccine based on recombinant attenuated Listeria monocytogenes and preparation method and application method thereof
  • KRAS high-expression cancer vaccine based on recombinant attenuated Listeria monocytogenes and preparation method and application method thereof

Examples

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preparation example Construction

[0059] The preparation method of recombinant plasmid pCW180 is as follows:

[0060] In step a, the Lm mpl gene carrying SpeI upstream and NotI downstream is inserted between the SpeI and NotI restriction sites of the plasmid pCW154 to obtain the first intermediate recombinant plasmid pCW160, the gene sequence of which is the sequence Shown in SEQ ID NO.4 in the list;

[0061] Step b, inserting the Lm orfBAldh gene carrying XbaI upstream and NotI downstream between the XbaI and NotI restriction sites of the first intermediate recombinant plasmid pCW160 to obtain the second intermediate recombinant plasmid pCW170, the second intermediate recombinant plasmid pCW170 The gene sequence is shown in SEQ ID NO.5 in the sequence listing;

[0062] Step c, digest plasmid pCW154 with NotI to obtain a gene with NotI restriction sites at both ends, and insert it into the NotI restriction site of the second intermediate recombinant plasmid pCW170 to obtain recombinant plasmid pCW180.

[006...

Embodiment 1

[0096] Embodiment 1. Preparation of vaccine bacteria LiΔactAplcB-KRAS;

[0097] (1) KRAS gene fragment synthesis;

[0098] Query the KRAS (G12D, G12V mutation) protein gene from NCBI, then optimize it according to the codons of Listeria, add a HindⅢ restriction site upstream, add a XhoⅠ restriction site downstream, and finally obtain the DNA sequence directly by synthesis (obtained in the form of cloning plasmid pUC57-KRAS provided by the company), (the sequence is shown in sequence 1 in the sequence listing).

[0099] (2) Construct targeting plasmid;

[0100] ① Construction of intermediate plasmid pCW203-KRAS

[0101] Plasmids pUC57-KRAS and pCW203 were extracted according to the instructions of the plasmids, and eluted with 30 μL Elution Buffer. pUC57-KRAS and pCW203 were double-digested with HindⅢ and XhoⅠ, restriction enzyme digestion system 20 μL: pUC57-KRAS or pCW2032 O Make up the system to 20 μL. Digest the plasmid in a water bath at 37°C for 1 hour, add 0.5 μL CIA...

Embodiment 2

[0138] Example 2: Preparation of the vaccine strain LmΔactAplcB-KRAS

[0139] (1) KRAS gene fragment synthesis; same as Example 1 (1).

[0140] (2) Construct targeting plasmid;

[0141] ①The construction of the intermediate plasmid pCW203-KRAS is the same as in Example 1(2)①

[0142] ②Construction of targeting plasmid

[0143] Plasmid pCW180 was extracted and eluted with 30 μL Elution Buffer. Mix plasmid pCW180 and intermediate plasmid pCW203-KRAS with restriction endonucleases BamHI and XhoI according to the system of (2) ① (replace HindIII enzyme with BamHI enzyme), carry out enzyme digestion and dephosphorylation, and gel recovery after electrophoresis The small fragment after pCW203-KRAS digestion is the insert fragment (244bp) and the pCW180 vector backbone (long fragment after digestion, about 8621bp in length). Mix according to the system of (2)①, connect and transform into Escherichia coli DH5α, smear on LA plate, perform PCR screening, BamHI and XhoI double enzyme...

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Abstract

The invention discloses a KRAS high-expression cancer vaccine based on recombinant attenuated Listeria monocytogenes and a preparation method and application method thereof. The cancer vaccine is bacterial liquid of an Lm delta actA / plcB-KRAS strain; the Lm delta actA / plcB-KRAS strain is a gene recombinant bacterium carrying a KRAS protein gene by taking the attenuated Listeria monocytogenes asa carrier; the attenuated Listeria monocytogenes is obtained by completely knocking out two virulence genes of actA and plcB from the Listeria monocytogenes; and the KRAS protein gene is a DNA sequence obtained by synthesizing a KRAS protein amino acid sequence after being optimized by a Listeria codon, adding a Hind III restriction enzyme cutting site at the upstream and adding an Xho I restriction enzyme cutting site at the downstream. According to the KRAS high-expression cancer vaccine disclosed by the invention, the attenuated Listeria monocytogenes is used as the carrier, and the specific immune response in a tumor microenvironment is effectively improved by utilizing the characteristic that the Listeria monocytogenes can uniquely grow in a host phagocyte and is a natural T cell immune activation adjuvant, so that the immune tolerance of an organism is broken, and further the development of tumors can be inhibited.

Description

technical field [0001] The invention relates to the field of biomedicine, and relates to the application of recombinant attenuated Listeria in the preparation of KRAS high-expression cancer therapeutic vaccines. Background technique [0002] Colorectal cancer is the third most common cancer worldwide, accounting for 10% of all cancers, with 1.09 million newly diagnosed cases and 551,000 deaths in 2018. Traditional surgery, radiotherapy and chemotherapy have limited therapeutic effects, and the five-year survival rate of colorectal cancer in the United States is 65%. Traditional surgical resection can be used to treat early colorectal cancer, and 50% of patients still die from distant metastasis of colorectal cancer. Although chemotherapy, radiotherapy and targeted therapy can prolong the life of patients, only 15% of patients with metastatic colorectal cancer can live beyond 5 years. In recent years, with the in-depth study of tumor immunity, new immunotherapy based on mob...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P35/00C12N1/21C12N15/12C12N15/74C12R1/01
CPCA61K39/001111A61P35/00C07K14/4748C12N15/74A61K2039/523A61K2039/522A61K2039/836Y02A50/30
Inventor 沈海浅王子豪沈浩
Owner IMMORNA (HANGZHOU) BIOTECHNOLOGY CO LTD
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