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Construction of immune-activated recombinant lactococcus lactis and application of immune-activated recombinant lactococcus lactis as tumor vaccines, immunologic adjuvants and like

A Lactococcus lactis, immune activation technology, applied in the fields of biomedicine and bioengineering, can solve the problem that immune active cells cannot fully exert specific immune cytotoxic effects, etc., to improve the tumor microenvironment, reduce immunosuppressive activity, and promote survival. and the effect of amplification

Pending Publication Date: 2022-01-18
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The failure of tumor therapy is often due to too many immunosuppressive factors in the tumor microenvironment, and too few immunocompetent cells such as DC and T cells in the tumor or cannot fully exert the specific immune cytotoxic effect

Method used

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  • Construction of immune-activated recombinant lactococcus lactis and application of immune-activated recombinant lactococcus lactis as tumor vaccines, immunologic adjuvants and like
  • Construction of immune-activated recombinant lactococcus lactis and application of immune-activated recombinant lactococcus lactis as tumor vaccines, immunologic adjuvants and like
  • Construction of immune-activated recombinant lactococcus lactis and application of immune-activated recombinant lactococcus lactis as tumor vaccines, immunologic adjuvants and like

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of Lactococcus lactis pNZ8148-Flt3L-OX40L:

[0034] According to SEQ ID NO.1, design and optimize the gene sequence of its coding gene as shown in SEQ ID NO.2, and design the corresponding restriction site.

[0035] The pNZ8148 vector plasmid was extracted, and the pNZ8148 vector plasmid and the target gene were digested with NcoI / HindIII; 37°C, 2h; then electrophoresed on 1% agarose gel, and the electrophoresis results were observed. The DNA recovery kit was used to recover the target gene fragment and the pNZ8148 vector plasmid double digestion product. The linearized fragment of the recovered pNZ8148 vector plasmid and the target gene fragment are ligated into a closed circular DNA molecule under the action of T4 DNA ligase through complementary cohesive ends to form the recombinant plasmid pNZ8148-Flt3L-OX40L.

[0036] The ligated recombinant plasmid was sent to be sequenced. The sequencing results were accurate and there were no frameshift mutations. ...

Embodiment 2

[0038] Construction of Lactococcus lactis pNZ8148-Flt3L-OX40L-cACMA:

[0039] According to SEQ ID NO.3, design and optimize the gene sequence of its coding gene as shown in SEQ ID NO.4, and design the corresponding enzyme cutting sites.

[0040] The pNZ8148 vector plasmid was extracted, and the pNZ8148 vector plasmid and the target gene were digested with NcoI / HindIII; 37°C, 2h; then electrophoresed on 1% agarose gel, and the electrophoresis results were observed. The DNA recovery kit was used to recover the target gene fragment and the pNZ8148 vector plasmid double digestion product. The linearized fragment of the recovered pNZ8148 vector plasmid and the target gene fragment are ligated into a closed circular DNA molecule through complementary cohesive ends under the action of T4 DNA ligase to form the recombinant plasmid pNZ8148-Flt3L-OX40L-cACMA.

[0041] The ligated recombinant plasmid was sent to be sequenced. The sequencing results were accurate and there were no frameshi...

Embodiment 3

[0043] Expression and detection of recombinant Lactococcus lactis pNZ8148-Flt3L-OX40L:

[0044] The recombinant plasmid pNZ8148-Flt3L-OX40L was transformed into lactic acid bacteria for protein expression and detection.

[0045] (1) Induced expression of pNZ8148-Flt3L-OX40L: The verified correct recombinant expression plasmid pNZ8148-Flt3L-OX40L was electrotransformed into Lactococcus NZ9000 to obtain recombinant lactic acid bacteria pNZ8148-Flt3L-OX40L, and the positive clone (pNZ8148-Flt3L- OX40L) was inoculated in a test tube of 5mL M17+0.5%glucose+10μg / mL chloramphenicol culture solution, cultured overnight at 30°C; the next day was inoculated in M17+0.5%glucose+10μg / mL chloramphenicol culture solution at a ratio of 1:100, Cultivate statically at 30°C until the OD600 of the bacteria is about 0.3-0.5; add Nisin to a final concentration of 10ng / mL, and culture at 30°C for 3-4 hours to induce the expression of the fusion protein.

[0046] (2) Detection of the expression of p...

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Abstract

The invention discloses construction of immune-activated recombinant lactococcus lactis and application of the immune-activated recombinant lactococcus lactis as tumor vaccines, immunologic adjuvants and the like, and belongs to the field of biomedicine and bioengineering. The two kinds of recombinant lactococcus lactis can express Flt3L-OX40L fusion protein at the corresponding positions of thalli. After successful construction, repeated experiments verify that the two recombinant lactococcus lactis vaccines can effectively activate dendritic cells (DC), and further can promote T cells to be differentiated into immune memory effector cells (TEM). Animal experiments indicate that injection of any immune-activated recombinant lactococcus lactis into an in-situ tumor has the effects of inhibiting the tumor and even enabling the tumor to completely disappear.

Description

technical field [0001] The present application relates to the fields of biomedicine and bioengineering, in particular, to a construction method and application of a recombinant Lactococcus lactis expressing Flt3L-OX40L fusion protein intracellularly or anchored to the cell wall. Background technique [0002] Tumor immunotherapy is by far the most eye-catching anti-tumor therapeutic field with the greatest clinical potential. In the past 10 years, tumor immunotherapy has made outstanding achievements in the following four aspects: (1) immune checkpoint regulators such as PD1 inhibitors; (2) adoptive immunotherapy of engineered T cells such as TCR-T, CAR-T, etc. ; (3) Antibody-drug conjugates (Antibody-drugconjugates, ADC) such as drugs formed by conjugating monastatin E (MMAE) and anti-CD30 human-mouse chimeric antibody Brentuximab; (4) Tumor vaccines such as new Antigen vaccines, in situ vaccines, etc. Nevertheless, there are still many challenges in the immunotherapy of s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/62C07K19/00A61K39/09A61K39/39A61P35/00C12R1/01
CPCC07K14/70575C07K14/52A61K39/09A61K39/39A61P35/00C07K2319/00C07K2319/035A61K2039/55594Y02A50/30
Inventor 刘宝瑞朱军梦
Owner NANJING DRUM TOWER HOSPITAL
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