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Dual-targeting fusion protein and encoding gene and application thereof

A fusion protein, dual-targeting technology, applied in the application field of fusion protein, dual-targeting fusion protein, and enhancing the immune activity of adenovirus vaccines, can solve problems such as toxic side effects, infection, etc.

Inactive Publication Date: 2015-11-04
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Type 5 adenovirus vectors infect host cells through the receptor (coxsackievirus and adenovirus receptor, CAR)-mediated pathway, but due to the low expression of the adenovirus receptor CAR on the surface of DCs, the titer of the virus must be increased to obtain sufficient infection efficiency; At the same time, most types of host cells express CAR on the surface, so when adenovirus vaccine is used to target DCs, adenovirus will also infect other host cells, which may cause some unexpected toxic side effects

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  • Dual-targeting fusion protein and encoding gene and application thereof
  • Dual-targeting fusion protein and encoding gene and application thereof
  • Dual-targeting fusion protein and encoding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0132] Example 1. Expression and Identification of the Dual Targeting Fusion Protein CFmDEC Mediating Type 5 Adenovirus Vector Infection of Dendritic Cells

[0133] The content of this embodiment includes:

[0134] 1) Obtain the coding gene of the dual targeting fusion protein CFmDEC capable of infecting dendritic cells with type 5 adenovirus vector;

[0135] 2) Construct a prokaryotic expression vector capable of expressing the fusion protein CFmDEC;

[0136] 3) express and purify the fusion protein CFmDEC in Escherichia coli;

[0137] 4) Determination of the concentration of the fusion protein CFmDEC by the Coomassie Brilliant Blue method;

[0138] 5) Western Blot identification of the fusion protein CFmDEC.

[0139] 1. Experimental materials and main equipment

[0140] 1. Primers, plasmids, strains

[0141] The primer fragments were synthesized by Beijing Zhongmei Taihe Biotechnology Co., Ltd.; Escherichia coli E.coli DH5α and BL21 (DE3) were purchased from Zhongmei Ta...

Embodiment 2

[0269] Example 2. In vitro functional verification of the dual-targeting fusion protein CFmDEC that mediates adenovirus infection of DC

[0270] The purpose of this example is to identify the biological activity of the dual-targeting fusion protein CFmDEC and verify its in vitro function. The specific contents include:

[0271] 1) Construct the prokaryotic expression vector pGEX-knob expressing type 5 adenovirus fiber noggin (Ad5-knob), express it in Escherichia coli BL21(De3) and purify it with GST agarose gel to obtain type 5 adenovirus fiber head knot protein Ad5-knob;

[0272] 2) Verify the specific binding ability of the dual-targeting fusion protein CFmDEC and the type 5 adenovirus fiber scallop protein Ad5-knob by enzyme-linked immunosorbent assay (ELISA);

[0273] 3) Isolate and cultivate mouse bone marrow-derived dendritic cells (mBMDC), extract cell membrane proteins, and verify the binding ability of the dual-targeting fusion protein CFmDEC and mBMDC membrane prote...

Embodiment 3

[0367] Example 3. Construction and Identification of Recombinant Adenovirus Vaccine Targeting Kidney Cancer-Associated Antigen G250

[0368] In this embodiment, double enzyme digestion was used to obtain the sig-tG250-Fc-GPI-IRES-GMCSF-B7.1 composite antigen (abbreviated as tG250FcGB or G250) gene fragment (its nucleotide sequence) from the eukaryotic expression plasmid pVAX-tG250FcGB As shown in sequence 11 in the sequence listing, bases 1-1131 from the 5' end are heterogeneous kidney cancer-associated antigen G250 (CAIX) genes, bases 1138-2520 from the 5' end are Fc-GPI, Bases 2568-3161 at the 5' end are the IRES sequence, and bases 3184-3597 at the 5' end are the granulocyte macrophage colony stimulating factor (GM-CSF) gene, starting at the 5' end The 3640-4428th base at the end is the co-stimulatory molecule B7.1 gene), and it is cloned into the adenovirus shuttle vector pDC316 to construct the shuttle plasmid pDC316-tG250FcGB (sequence 12); according to the AdMax adenovi...

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Abstract

The present invention discloses dual-targeting fusion protein CFmDEC capable of mediating a type 5 adenovirus vector to be specifically bound with a surface molecule DEC205 of a dendritic cell; and by utilizing a gene fragment sig-tG250-Fc-IRES-GM-CSF-B7.1(25) of a multi-targeted anti-renal cancer compound antigen, an anti-renal cell carcinoma adenovirus vaccine-Ad5-tG250FcGB is constructed with a type 5 replication-deficient adenovirus as a vector. The dual-targeting fusion protein CFmDEC is capable of enhancing the antitumor effect of the renal cell cancer adenovirus vaccine Ad5-tG250FcGB, is effectively combined with the adenovirus, improves the DCs-targeted ability of the adenovirus vaccine, and can induce higher specific antitumor immune response in a human body and suppress the growth of renal cell cancer.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a fusion protein, in particular to a dual-targeting fusion protein (CFmDEC) capable of mediating type 5 adenovirus vectors specifically binding to the surface molecule DEC205 of dendritic cells (DCs) and its encoding Gene and its application in enhancing the immune activity of adenovirus vaccine. Background technique [0002] 1. Research progress of tumor gene vaccines [0003] The concept of tumor immunotherapy is based on the fact that the human immune system can recognize tumor cells from normal cells, thereby affecting the growth of tumor cells. Evidence shows that the tolerance of the immune system to tumor cells is incomplete, and the intensity and scope of the spontaneous immune response to tumor cells are correlated with tumor prognosis and survival time. However, the anti-tumor immune response is usually affected by the immunosuppressive response in the tumor microenvir...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N1/15C12N1/19A61K39/39A61P35/00
Inventor 于继云阎瑾琦王宇田仁礼殷小涛武帅朱晓明王籽橙杜芝燕
Owner ACADEMY OF MILITARY MEDICAL SCI
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