Cervical cancer therapeutic vaccine based on recombinant attenuated Listeria monocytogenes

A Listeria monocytogenes, therapeutic vaccine technology, applied in the biological field, can solve problems such as defects and low immunogenicity

Active Publication Date: 2018-12-14
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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  • Cervical cancer therapeutic vaccine based on recombinant attenuated Listeria monocytogenes
  • Cervical cancer therapeutic vaccine based on recombinant attenuated Listeria monocytogenes
  • Cervical cancer therapeutic vaccine based on recombinant attenuated Listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Preparation of vaccine bacteria LmΔactAplcB-HPV

[0067] (1) HPV16E6E7 gene fragment synthesis

[0068] 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 correspondingly according to Listeria codons, A HindⅢ restriction site was added upstream, and an XhoI restriction site was added downstream. 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 (such as SEQ ID NO.1 shown).

[0069] (2) Construction of targeting plasmid

[0070] ① Construction of intermediate plasmid pCW203-E6E7

[0071] Plasmids pUC57-E6E7 and pCW203 were extracted according to the instructions of the plasmids, and eluted with 30 μL Elution Buffer.

[0072] pUC57-E6E7 and pCW203 were double-dig...

Embodiment 2

[0096] Example 2: Evaluation of the therapeutic effect of vaccine strains on cervical cancer

[0097] (1) TC-1 tumor cell culture and establishment of cervical cancer model

[0098] 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). Data were statistically analyzed using SPSS software. The t-test was used to compare the means of the two...

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Abstract

The invention relates to a cervical cancer therapeutic vaccine based on recombinant attenuated Listeria monocytogenes. Attenuated Listeria monocytogenes (hereinafter referred to as Lm) is used as a vector, and the unique ability of Listeria to grow in a host phagocytic cell is the characteristic of a natural T cell immune activation adjuvant, and is used to effectively improve the specific immuneresponse in a tumor microenvironment and break the immune tolerance of bodies, so persistent viral infection of the bodies is removed, and good therapeutic effects of clearing focus and restraining tumor development are achieved. The cervical cancer therapeutic vaccine makes up for the shortcoming of low immunogenicity of nucleic acid and protein peptide vaccines, and the attenuated Listeria reserves the intracellular growth and antigen presentation completion characteristics of an original strain, and has a high safety.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a cervical cancer therapeutic vaccine based on recombinant attenuated Listeria monocytogenes. 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 the 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 continuously high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K39/12A61P35/00A61P31/20C12N1/21C12N15/74C12R1/01
CPCA61K39/12A61K39/39A61K2039/523A61K2039/53A61K2039/55594A61K2039/585A61P31/20A61P35/00C07K14/005C07K2319/00C12N15/74C12N2710/20022C12N2710/20034
Inventor 汪川沈海浅
Owner 南京颂悦生物科技有限公司
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