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sHSPs recombinant invasive lactobacillus plantarum live vector DNA vaccine

A technology of Lactobacillus plantarum and live carrier vaccines, applied in the direction of recombinant DNA technology, DNA/RNA vaccination, vaccines, etc., can solve the problems of difficult immune response, etc., and achieve the effects of easy promotion, high safety and convenient use

Pending Publication Date: 2021-08-06
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, the antigens used in recombinant lactic acid bacteria tumor vaccines were tumor autoantigens, which were prone to immune tolerance and difficult to induce a strong immune response. Trichinella sHSPs are heterologous antigens that can break immune tolerance and induce anti-tumor immunity

Method used

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  • sHSPs recombinant invasive lactobacillus plantarum live vector DNA vaccine
  • sHSPs recombinant invasive lactobacillus plantarum live vector DNA vaccine
  • sHSPs recombinant invasive lactobacillus plantarum live vector DNA vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] SHSPS gene amplification:

[0039] According to the SHSPS gene DNA sequence of the spiillase SHSPS gene:

[0040] Upstream primers: 5'-gdomccccccatg Gactacaaagacgatgacgacaaaaa AtggcctTgactgcgtggTataAT-3 ',

[0041] Downstream primers: 5'ctcgagtta Atggtgatgtggtgatg TctCTCTGAGCAGCGGAGCG-3 ';

[0042] The enzyme disaster is: GGATCC ( Bamh I ); Ctcgag Xho I ); The upstream primer is bold to kozak sequence, and the underscore is a FLAG sequence; and the downstream primer is bolded to terminate the codon sequence, and the underscore is HIS sequence;

[0043] PCR amplification of the spiper SHSPS gene, the colloated recovery was cloned to the PMD18-T vector and was subjected to bacterial liquid PCR, sequencing identification and named PMD18-T-SHSPS.

Embodiment 2

[0045] Construction of SHSPS recombinant invasive plant Lactobacillus:

[0046] use Bamh I , Xho I Extract and recover PMD18-T-SHSPS, with Bamh I , Xho I Enzyme digested PVALAC carrier connection, ligation product conversion E.COLI The recombinant plasmid was screened after TG-1 perceptual cells, sequencing, and the recombinant plasmid was named pVALAC-SHSPS after the enzyme digestion was identified. Recombinant plasmid pVALAC-SHSPS transfected 293T cells, Western blotting verification expression (see figure 1). After verifying, 5 μL of empty load plasmid PVALAC, recombinant plasmid pVALAC-SHSPS is mixed with 100 μL of pre-cooling sensitudinal NC8 (CCUG 61730), added to a pre-cooling electric shock cup, ice bath 5 min, in the electroactor Electric shock (2.5 kV, 6 ms), ice bath 5 min, remove liquid transfer to 850 μL MRS medium (containing 0.5 mol / L sucrose), 30 ° C water bath 3 h, coated to cm + (10 μg / ml) MRS solid medium, anaerobic culture at 37 ° C to pick out single colon...

Embodiment 3

[0048] Recombinant invasive plant Lactobacillus NC8-SHSPS planting capacity detection

[0049] C57BL / 6 mouse is immunized NC8-SHSPS, setting blank control and NC8-PVALAC control, first 3 D, then immunized after 14 days, 3 days, each time 9 CFU / 200 μL. 2 free 7 days, sterile separation into the intestines, take some content, PBS double compared to dilution, apply 50 μL of dilution sample to CM + MRS solid medium. At 37 ° C Anaerobic culture 24 h, calculated the number of colonies on solid medium. The recombinant plant Lactobacillus NC8-SHSPS was immunized to mice, and the intestinal can be detected, and the content of recombinant plant NC8-SHSPs in 0.2 g intestinal content is 9.28 × 10 5 CFU (see figure 2 ).

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Abstract

The invention aims to provide an sHSPs recombinant invasive lactobacillus plantarum live vector DNA vaccine. Lactobacillus is selected as a vector to construct the recombinant invasive lactobacillus plantarum. The lactobacillus plantarum has immunoregulation ability and carries tumor antigen to inhibit tumor growth, and trichina sHSPs as a heteroantigen can break immune tolerance, such that obvious anti-tumor immunity is generated. The lactobacillus plantarum carries the related antigen gene sHSPs so as to provide non-specific immunoregulation ability of the lactobacillus, and the sHSPs can be delivered to a host cell to express the related antigen sHSPs so as to induce a specific immune response. The recombinant lactobacillus plantarum carrying the trichina sHSPs gene exert non-specific immunoregulation ability of the lactobacillus and can also induce specific anti-tumor immunity. Meanwhile, as a probiotic, the lactobacillus has high safety, can be orally taken, and is convenient to use and easy to popularize.

Description

Technical field [0001] The present invention discloses a SHSPS recombinant intrusion plant Lactobacillus live DNA vaccine, is a spiral and Lewis lung cancer-related antigen gene SHSPS recombinant invasive plant lactoforma live carrier DNA vaccine, which can be used for the prevention and treatment of LEWIS lung cancer, a biological Medical field. Background technique [0002] Lung cancer is the highest mortality cancer, causing serious economic burden. In recent years, the incidence of lung cancer has increased increasingly, and it is a young trend. At present, there is no ideal preventional treatment of lung cancer, and new therapies is of great significance. The vaccine is divided into therapeutic vaccine and preventive vaccines, which is an important means of prevention and treatment. R & D Cancer Vaccine is one of the hotspots of cancer research. [0003] In recent years, the study found that bacterial live carrier vaccines can be used for tumor prevention and exhibit signifi...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/74C12N1/21A61K39/00A61P35/00C12R1/25
CPCC07K14/4354C12N15/74A61K39/0003A61P35/00A61K2039/53A61K2039/523
Inventor 张西臣岳涛涛张楠宫鹏涛李建华刘少雄王晓岑李新程淑琴
Owner JILIN UNIV
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