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Preparation and application of oral tumor vaccine with attenuated salmonella typhimurium as vector

A technology of salmonella and tumor vaccine, which is applied in the field of preparation of oral tumor vaccine

Inactive Publication Date: 2014-02-05
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Minigene vaccine can stimulate high level of CD8+ T cell response, and AIDA-I system also has advantages in stimulating CD4+ T cell response, but there is no AIDA-I system -A report on the integration of I surface display system and microgene vaccine in the same tumor vaccine vector

Method used

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  • Preparation and application of oral tumor vaccine with attenuated salmonella typhimurium as vector
  • Preparation and application of oral tumor vaccine with attenuated salmonella typhimurium as vector
  • Preparation and application of oral tumor vaccine with attenuated salmonella typhimurium as vector

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Construction of Oral Tumor Vaccine Vector Integrating Minigene and AIDA-1 Delivery System

[0041] attached figure 1 Schematic diagram of the construction of an oral tumor vaccine vector integrating Wegene and AIDA-1 delivery system, the specific steps are:

[0042] 1. Construction of PMK90 vector: Take Escherichia coli E393, boil it for 10 minutes as a PCR template, construct the plasmid pMK90 carrying the original expression of AIDA-1, and digest it with KpnI and XbalI at the C-terminal primer AgeI site of AIDA-I For the plasmid, the large fragment of the vector is recovered for use;

[0043] 2. Using the cDNA of B16 cells as a template, use primers Seq NO.1 and SeqNO.2 to amplify the Melan-A gene; use KpnI and XbalI to double digest, recover the product and connect it to the large fragment of the recovered vector; Transform the ligation product into DH10B competent bacteria, pick the bacteria and identify the positive plasmid; take the plasmid for furth...

Embodiment 2

[0048] Embodiment two : Tumor-preventive effect of an oral tumor vaccine vectored with attenuated Salmonella

[0049] (1) Preparation of oral tumor vaccine with attenuated Salmonella as carrier:

[0050] Take 10mL of Salmonella in the logarithmic growth phase, centrifuge at 1500g-2000g for 10min at 4°C, discard the supernatant; use 20mL ice-cold (0-4°C) WB buffer (sterile ultrapure water containing 10% glycerol) Resuspend the pellet, centrifuge at 1500g-2000g for 10min at 4°C, discard the supernatant; resuspend the pellet with 10mL ice-cold (0-4°C) WB buffer, centrifuge at 1500g-2000g for 10min at 4°C, discard For the supernatant, resuspend the pellet with 1mL of ice-cold (0-4°C) WB buffer; take 50uL of the suspension and add it to a 0.2cm electric cup in an ice bath, then add 20pg-0.1mg of the plasmid pV prepared in Example 1, Mix gently; put the electro-spinning cup into the Bio-Rad Gene Pulser instrument, and conduct electric shock at 2.5 kV, 25 mF, 200 V; quickly take o...

Embodiment 3

[0053] Embodiment Three : Oral tumor vaccine vectored with attenuated Salmonella induces CD4+ T cell and CD8+ T cell antitumor immune responses

[0054] The mice in Embodiment 2 were sacrificed 18 days after tumor inoculation, the spleen and tumor cells of the mice were collected, and the responses of CD4+ T cells and CD8+ T cells were analyzed by cell counting and flow cytometry.

[0055] attached image 3This is the effect of the above oral tumor vaccine on the immune response of T cells in the spleen. It can be seen that the IFN-γ-positive CD4+ T cells in the spleen of mice inoculated with pV-carrying SL7207 (Vaccine) group were significantly higher than those in other groups, and the IFN-γ-positive The number of CD8+ T cells is also significantly increased; indicating that oral administration of the oral tumor vaccine disclosed in the present invention can promote the immune response of CD4+ T cells and CD8+ T cells.

[0056] attached Figure 4 The promotional effect o...

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Abstract

The invention discloses a preparation and application of an oral tumor vaccine with attenuated salmonella typhimurium as a vector. The preparation of the vaccine comprises the following steps: (1) firstly, establishing a recombined vaccine vector integrating a micro gene with a self-transfer system through a molecular biological method; and (2) electrically transforming the recombined vaccine vector created in the step (1) into attenuated salmonella typhimurium SL7207 to establish the recombinant oral tumor vaccine. According to the preparation disclosed by the invention, the micro gene and the self-transfer system are integrated onto one vector for the first time, the salmonella typhimurium is electrically transformed to obtain the oral tumor vaccine, and the immune prevention effect of the vaccine on tumors is verified by a mouse tumor model, so that the oral tumor vaccine can be applied in the field of preparing immune preventive medicines for tumors.

Description

technical field [0001] The invention relates to the preparation of a mouse tumor vaccine, in particular to the preparation and application of an oral tumor vaccine based on a minigene and an autotransport system with attenuated Salmonella as a carrier. Background technique [0002] The immune system mainly kills tumor cells through CD8+ T cells mediating cytotoxic T lymphocyte (cytotoxicity T lymphocyte, CTL) effect. More and more studies have shown that CD4+ T cells also play a key role in anti-tumor immunity. CD4+ T cells can control the generation of memory CD8+ T cells through a TRAIL-dependent mechanism, and tumor-specific CD4+ T cells can maintain the immune memory of CD8+ T cells and increase the tumor infiltration level of CD8+ T cells. However, it is often necessary to use multiple vaccines in combination to induce high levels of CD4+ and CD8+ T cell responses; moreover, the currently designed vaccines often require multiple immunizations by injection, which limits...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/00A61P35/00C12N15/74C12N1/21C12R1/42
Inventor 赵李祥梅雨刘海燕
Owner SUZHOU UNIV
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