Recombinant vaccine and application thereof

A technology of vaccines and recombinant vectors, applied in vaccines, applications, recombinant DNA technology, etc., can solve the problems of sPD1 synergistic anti-tumor effect disappearing, no anti-tumor effect, etc.

Inactive Publication Date: 2018-04-24
CHANGCHUN BCHT BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results showed that the combined therapy group had an anti-tumor effect of 90%, which had a good synergistic effect compared with the single HSVtk therapy; while the sPD1 gene therapy group alone had no anti-tumor effect; when CD8-positive T cells in mice were deleted After that, the synergistic antitumor effect of sPD1 disappeared

Method used

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  • Recombinant vaccine and application thereof
  • Recombinant vaccine and application thereof
  • Recombinant vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Embodiment 1: Construction of recombinant DNA vector vaccine sPD1 / MS

[0131] 1.1 tPA / sPD1 gene synthesis and T Easy-tPA / sPD1 construction

[0132] According to GeneBank number NM_005018.2 gene synthesis of human PD-1 extracellular segment sequence, the 5' end of the gene sequence is connected with a 69bp tPA signal peptide sequence (see SEQ ID NO: 10), the purpose of introducing the tPA signal peptide is to improve the fusion protein The ability of sPD1 / MS to secrete and express extracellularly. Thereafter, PCR technology was used to introduce restriction sites at both ends of the tPA / sPD1 gene, and the PCR reaction system was 50 μl, including 0.1 μg of the tPA / sPD1 DNA template described in SEQ ID NO: 1, primers SEQ ID NO: 12 and SEQ ID NO: 13 Final concentration 20pmol, 5μl Ex Taq Buffer (10×), 4μl dNTP Mixture (2.5mM each), 1.25U ExTaq enzyme (Takara); 35 cycle amplification reactions. Subsequently, the PCR product recovered from the gel was connected to the pGEM...

Embodiment 2

[0141] Embodiment 2: Preparation of recombinant protein vaccine sPD1 / MS

[0142] Protein vaccines are another common form of tumor vaccines. Different from DNA vaccines or virus vector vaccines, protein vaccines enter cells in the form of antigenic proteins and can directly activate dendritic cells. At the same time, because the protein form cannot be integrated into the host cell genome, it has Higher security. In this example, the prokaryotic expression vector pET28a-sPD1 / MS was constructed to induce the expression of the fusion protein vaccine (sPD1 / MS). The specific construction process is as follows:

[0143] The prokaryotic expression plasmid pET28a-sPD1 / MS was constructed by homologous recombination: the pET28a plasmid (see image 3 -a) After double digestion with Nde I and Xho I, a 5289 bp fragment of the vector sequence was recovered from the gel. The sequence of the sPD1 / MS cistron in CpDV-IL2-sPD1 / MS was amplified by PCR, and the 5' end of the primer had a sequenc...

Embodiment 3

[0148] Embodiment 3: Construction of recombinant viral vector vaccine sPD1 / MS

[0149] 3.1 Obtaining of recombinant poxvirus vaccine rMVA-sPD1 / MS

[0150] The application of Modified Vaccinia Ankara (MVA) co-carried with tumor antigens for immunotherapy of tumors is relatively safe. The shuttle plasmid of MVA is pSC11, which has multiple cloning restriction sites that can be inserted into foreign genes. In this example, the sPD1 gene was inserted into the previously constructed plasmid pSC11-MS (see the invention patent with the patent number ZL200910252427.X), To construct pSC11-sPD1 / MS, the thymidine kinase left and right arms (TKL, TKR) on the vector can be homologously recombined with MVA, and at the same time, the blue spot screening of recombinant MVA (rMVA) can be carried out through the lacZ gene on the vector.

[0151] The specific method for constructing the pSC11-sPD1 / MS shuttle plasmid is first to carry out a PCR reaction with primers SEQ ID NO: 16 and SEQ ID NO: ...

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Abstract

The invention relates to the technical field of biotech drugs, in particular to a recombinant vaccine and application thereof. The invention provides a molecular combination which comprises soluble PD-1, MUC1 and Survivin. The vaccine is prepared by the molecular combination and has good immunogenicity and antitumor activity. The vaccine provided by the invention can generate specific antibody response and specific cell immune response aiming at the MUC1 and the Survivin. Compared with a DNA vaccine CpDV-IL2-MS, CpDV-IL2-sPD1/MS has a more obvious antitumor effect in tumor-bearing mice. The invention further provides a joint program of a therapeutic vaccine and a chemotherapeutic drug.

Description

technical field [0001] The invention relates to the technical field of biotechnology drugs, in particular to recombinant vaccines and applications thereof. Background technique [0002] Cancer or malignant tumors are killers that threaten human health. The causes of tumors are complex, and we still lack a deep understanding of their specific formation mechanisms. Recent studies have shown that there is a cyclic interaction between the body's immune system and tumor cells, that is, the immune system can recognize cancer cells with "non-self" components, and cancer cells can also escape the monitoring and attack of the body's immune system through various ways, causing patients Diffusion and metastasis of tumor cells in vivo. The molecular mechanisms of tumor immune escape include: downregulation or loss of tumor antigen expression, secretion of soluble cytokines with immunosuppressive function by tumor cells, recruitment of immunosuppressive lymphocytes by the tumor microenv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/39A61K39/235A61K39/275A61K31/555A61P35/00C12N15/85C12N15/861C12N15/863C07K19/00
CPCA61K31/555A61K39/0011A61K39/12A61K39/39A61K2039/5154A61K2039/53A61K2039/55505A61K2039/55522A61K2039/55533A61K2039/575C07K14/4727C07K14/4747C07K14/70521C07K2319/02C07K2319/32C12N15/85C12N15/86C12N2710/10234C12N2710/10343C12N2710/24034C12N2710/24043A61K2300/00
Inventor 孔维张海红于湘晖陆臻桢刘晨露徐平耿飞谢雨郭倩倩
Owner CHANGCHUN BCHT BIOTECH
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