Application of recombinant attenuated Listeria in preparation of therapeutic vaccine for cervical cancer

A therapeutic vaccine and Listeria technology are applied in the application field of recombinant attenuated Listeria in the preparation of cervical cancer therapeutic vaccines, and can solve problems such as defects and low immunogenicity.

Active Publication Date: 2018-12-18
南京颂悦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key to its success is to induce a sufficient immune response, but the current nucleic acid and protein peptide vaccines have low immunogenicity and obvious defects

Method used

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  • Application of recombinant attenuated Listeria in preparation of therapeutic vaccine for cervical cancer
  • Application of recombinant attenuated Listeria in preparation of therapeutic vaccine for cervical cancer
  • Application of recombinant attenuated Listeria in preparation of therapeutic vaccine for cervical cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1. Preparation of vaccine bacteria LiΔactAplcB-E6E7

[0101] (1) HPV16 E6E7 gene fragment synthesis

[0102] Query HPV 16 E6 protein and E7 protein genes from NCBI, use online software to analyze and predict their dominant T cell antigen epitopes, select and combine them into fusion antigen epitope peptide genes, and then optimize according to Listeria codons, And a HindⅢ restriction site was added upstream, and an XhoI restriction site was added downstream, and finally the DNA sequence was directly obtained by synthesis (obtained in the form of the cloning plasmid pUC57-E6E7 provided by the company), and the fragment length was 866bp (the sequence is shown in the sequence table sequence 1 shown).

[0103] (2) Construction of targeting plasmid

[0104] ① Construction of intermediate plasmid pCW203-E6E7

[0105] Plasmids pUC57-E6E7 and pCW203 were extracted according to the instructions of the plasmids, and eluted with 30 μL Elution Buffer.

[0106] pUC57-E6E...

Embodiment 2

[0130] Embodiment 2: the preparation of vaccine bacterium LmΔactAplcB-E6E7

[0131](1) HPV16 E6E7 gene fragment synthesis

[0132] Same as Example 1(1)

[0133] (2) Construction of targeting plasmid

[0134] ①The construction of the intermediate plasmid pCW203-E6E7 is the same as in Example 1(2)①

[0135] ②Construction of targeting plasmid

[0136] Plasmid pCW180 was extracted and eluted with 30 μL Elution Buffer. Mix plasmid pCW180 and intermediate plasmid pCW203-E6E7 with restriction endonucleases BamHI and XhoI respectively 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 digestion of pCW203-E6E7 is the insert fragment (931bp) and the backbone of the pCW180 vector (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, perfo...

Embodiment 3

[0150] Example 3: Evaluation of the therapeutic effect of vaccine strains on cervical cancer

[0151] (1) TC-1 tumor cell culture and establishment of cervical cancer model

[0152] Use RPMI1640 Medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in 5% CO 2 Cultivate TC-1 cells in an incubator until the density reaches about 80%, rinse the cells with Hank’s solution for 2 to 3 times, digest with trypsin and resuspend with PBS for counting, and prepare the cells to a concentration of 1×10 6 100 μl of cell suspension was subcutaneously injected into the right abdomen of 6-8 week-old C57BL / 6 female mice to establish a mouse HPV-infected tumor model, and a macroscopic tumor was formed in about 7 days. Tumor volume according to the formula 1 / 2×(a×b 2 ) calculation (a, b are the longest diameter and shortest diameter of the tumor, respectively). Statistical analysis of data was performed using SPSS software. The t-test was used to compare the means ...

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Abstract

The invention relates to application of recombinant attenuated Listeria in preparation of a therapeutic vaccine for cervical cancer. Attenuated Listeria monocytogenes (hereinafter referred to as Lm) and attenuated Listeria ivanovii (hereinafter referred to as Li) are used as vectors, by use of the unique characteristic that the Listeria can grow in phagocytose cells in a host, and is a natural T cell immune activation adjuvant, specific immune response in tumor microenvironment can be effectively enhanced, body's immune tolerance can be broken, body's persistent viral infection can be eliminated, and good treatment results of clearing focuses and curbing tumor development can be achieved. The Listeria has the advantages that shortcomings of low immunogenicity of nucleic acid and protein peptide vaccines can be made up, the attenuated Listeria retains the characteristic that an attenuated original strain can grow intracellularly to complete antigen presentation, and meanwhile the attenuated Listeria has higher safety.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the application of recombinant attenuated Listeria in the preparation of cervical cancer therapeutic vaccine. Background technique [0002] Cervical cancer is a serious threat to the health of women all over the world. It is a gynecological malignancy with a mortality rate second only to breast cancer. Studies have shown that HPV is a major risk factor for cervical cancer, and more than 90% of cervical cancer patients are infected with high-risk HPV types, among which types 16 and 18 are the main ones. 90% of cervical cancer is cervical squamous cell carcinoma, and its main pathogenic type is HPV16. The main carcinogenic factors of HPV are E6 and E7 proteins. E6 destroys normal cell cycle regulation by inhibiting the activity of tumor suppressor p53 and promotes cell immortalization; E7 protein loses its tumor suppressor effect by binding and degrading pRb protein. E6 and E7 are conti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61P31/20A61P35/00C12N15/74C12N15/62
CPCA61K39/12A61P31/20A61P35/00C12N15/62C12N15/74C07K14/005C12N2710/20022C12N2710/20034A61K2039/522A61K2039/523C07K2319/00
Inventor 汪川沈海浅
Owner 南京颂悦生物科技有限公司
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