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Tumor nucleic acid vaccine based on tissue factor, preparation method and application thereof

A tumor nucleic acid vaccine and tissue factor technology, applied in gene therapy, anti-tumor drugs, medical preparations containing active ingredients, etc., can solve problems such as allergic reactions, difficult processes, and complex protein structures

Active Publication Date: 2016-09-14
JIANGSU HIGH WIT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, they use the whole TF protein as the target and use antibodies or FVII for treatment, which has the disadvantages of complex protein structure, difficult process, and easy triggering of allergic reactions in the body.

Method used

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  • Tumor nucleic acid vaccine based on tissue factor, preparation method and application thereof
  • Tumor nucleic acid vaccine based on tissue factor, preparation method and application thereof
  • Tumor nucleic acid vaccine based on tissue factor, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Construction of eukaryotic expression vector pCR-X8-HBcΔ-TF

[0021] 1.1 Construction of eukaryotic expression vector pCR-X8-HBcΔ-TF

[0022] 1.1.1 Cloning of TF target gene

[0023] Gene cloning of TF target fragment

[0024] Design upstream and downstream primers as templates for each other, and obtain target fragments by PCR

[0025] TF

[0026] Upstream primer TF-up: 5'- T GGT ACC GGT GAC TGG AAA TCC AAA TGC TTC TAC ACTACT GAC A-3' (SEQ ID NO: 2)

[0027] Downstream primer TF-down: 5'- AAA AAGC TTA ACC GGT TTC GTC AGT CAA GTC GTATTC AGT GTC AGT AGT GTA GAA GCA T-3' (SEQ ID NO: 3)

[0028] Both upstream and downstream primers contain restriction endonuclease AgeI sites (indicated by the shaded part)

[0029] PCR reaction system:

[0030] 10×PFU PCR buffer 10 µl

[0031] TF cDNA 2 μl

[0032] 10×PFU PCR buffer 10 μl

[0033] Upstream primer 8 μl

[0034] Downstream primer 8 μl

[0035] dNTP 8 μl

[0036] PFU DNA Polymerase 1 μl

[0037] wxya 2...

Embodiment 2

[0067] Example 2: ELISA detection and Western blot detection of TF antibody

[0068] 2.1 Materials

[0069] 2.1.1 Experimental animals

[0070] C57 / BL6 pure line mice, female, 6-8 weeks old, with an average body weight of 16.8 g before immunization, were purchased from the Experimental Animal Center of Yangzhou University.

[0071] experimental drug

[0072] Recombinant nucleic acid vaccine HBcΔ-TF.

[0073] Materials and Reagents

[0074] Purified recombinant proteins VEGF-TFh, VEGF-TFM, VEGF-PVH, VEGF-PVM. The specific test operation is the same as before.

[0075] Materials and Reagents

[0076] Purified recombinant proteins VEGF-TF, VEGF-PV. The specific test operation is the same as before.

[0077] method

[0078] 2.2.1 Immunization program

[0079] C57BL / 6J female mice were selected and divided into random groups, 10 mice in each group, including normal saline control group, plasmid vector control group and pCR-X8-HBcΔ-TF group. Nucleic acid vaccine or contro...

Embodiment 3

[0088] Example 3: Research on anti-tumor effect of recombinant TF subunit epitope nucleic acid vaccine

[0089] 3.1 Tumor cell culture and transplantation tumor model preparation

[0090] 3.1.1 Materials

[0091] B16-F10 was purchased from the Institute of Cells, Chinese Academy of Sciences, RPMI1640 and DMEM medium were purchased from JIBCOL, and C57 / BL6 mice were purchased from the Comparative Medicine Center of Yangzhou University.

[0092] Cell Culture and Transplantation

[0093] B16-F10 is a highly metastatic melanoma strain with a C57 / BL6 background, and it is also an adherent growth cell. The culture medium is DMEM medium containing 10% calf serum, and it is inoculated in a culture flask. 5%CO 2 When the cells grow into a continuous monolayer under certain conditions, carefully pour off the medium, gently wash with D-HANKS solution, pour off the D-HANKS solution, and add 0.125% trypsin to digest. Pour off the trypsin, immediately add 10% calf serum 1640 medium to s...

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Abstract

The invention discloses a tumor nucleic acid vaccine based on tissue factor, which uses a plasmid as a carrier, inserts a DNA sequence encoding HBc into the plasmid, and inserts a DNA sequence encoding a human TF target epitope polypeptide into the sequence, the human TF target The epitope polypeptide sequence is shown in SEQ ID NO: 1, and the polypeptide is amino acid 74-94 of human TF; the non-coding region of the plasmid vector contains the ISS sequence. The preparation method and application of the tumor nucleic acid vaccine are also disclosed. In the present invention, by means of genetic engineering, a segment of epitope target sequence selected from TF is presented as an antigenic epitope to the remodeled hepatitis B core antigen to construct an antigenic epitope nucleic acid vaccine, which is given to the body by intramuscular injection to promote Host cells express polypeptides containing TF target epitopes to stimulate the body to produce active immunity, thereby obtaining preventive and therapeutic effects on tumors.

Description

technical field [0001] The invention relates to a nucleic acid vaccine, a preparation method and its application, especially a tumor nucleic acid vaccine, a preparation method and its application. Background technique [0002] The coagulation function of tissue factor (TF) is well known. After it combines with activated coagulation factor VII (activated factor VII, F VIIa), it forms a complex of TFPF VIIa to initiate the extrinsic coagulation pathway; it can also activate coagulation factor XI, Activate the intrinsic coagulation system. It is one of the most active procoagulant substances in the body, and plays an important role in physiological hemostasis, coagulation process and various thromboembolic diseases. However, as the only transmembrane glycoprotein expressed on the cell surface in the blood coagulation system, whether TF has receptor properties and non-coagulation function has gradually become the focus of many scholars. Recent studies have shown that TF has th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/29C12N15/70A61P35/00
Inventor 刘文涛吴雪丰徐礼友杨政
Owner JIANGSU HIGH WIT BIOTECH CO LTD
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