Cervical cancer therapeutic vaccine based on recombinant attenuating sheep listeria ivanovii

A therapeutic vaccine, the technology of Listeria, is applied in the field of cervical cancer therapeutic vaccine, which can solve the problems of defects and low immunogenicity

Active Publication Date: 2018-12-07
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 n

Method used

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  • Cervical cancer therapeutic vaccine based on recombinant attenuating sheep listeria ivanovii
  • Cervical cancer therapeutic vaccine based on recombinant attenuating sheep listeria ivanovii
  • Cervical cancer therapeutic vaccine based on recombinant attenuating sheep listeria ivanovii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Preparation of vaccine bacteria LiΔactAplcB-E6E7

[0061] (1) HPV16 E6E7 gene fragment synthesis

[0062] 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 Xho I 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 (such as SEQ ID NO. 1).

[0063] (2) Construction of targeting plasmid

[0064] ① Construction of intermediate plasmid pCW203-E6E7

[0065] Plasmids pUC57-E6E7 and pCW203 were extracted according to the instructions of the plasmids, and eluted with 30 μL Elution Buffer.

[0066] pUC57-E6E7 and pCW203 were digested with HindI...

Embodiment 2

[0090] Example 2: Evaluation of the therapeutic effect of vaccine strains on cervical cancer

[0091] (1) TC-1 tumor cell culture and establishment of cervical cancer model

[0092] 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 a cervical cancer therapeutic vaccine based on recombinant attenuating sheep listeria ivanovii. By taking the attenuating sheep listeria ivanovii as a vector, specific immuneresponse in a tumor microenvironment is improved effectively and immune tolerance of a body is broken by means of a unique characteristic that the listeria ivanovii can grow in a main phagocyte and isa natural T cell immune activation adjuvant, so that continuous virus infection of the body is eliminated and the good therapeutic effect of eliminating the focus and inhibiting tumor development isachieved. The vaccine has the advantages that the defect that nucleic acid and protein polypeptide vaccines are relatively low in immunogenicity is made up, and the attenuating sheep listeria ivanoviialso has high safety while retaining the characteristic that an original strain can grow in cells to achieve antigen presentation.

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 ovis. 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 continuously highly expressed ...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61K39/12A61P35/00A61P31/20C12N1/21C12N15/74C12R1/01
CPCA61K39/12A61K39/39A61P31/20A61P35/00C12N15/74C07K14/195A61K2039/523A61K2039/53A61K2039/55594A61K2039/585C12N2710/20034
Inventor 汪川沈海浅
Owner 南京颂悦生物科技有限公司
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