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A tumor cell-specific effector gene expression vector initiated by NF-κB, its expression product and application

A technology of effector gene and gene expression, applied in the direction of introducing foreign genetic material, carrier, anti-tumor drugs, etc. using vectors, can solve the problems of side effects, excessive inhibition of NF-κB activity, etc.

Active Publication Date: 2020-07-31
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Introducing NF-κB inhibitors to cells, because the amount cannot be controlled, often leads to excessive inhibition of NF-κB activity, resulting in serious side effects

Method used

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  • A tumor cell-specific effector gene expression vector initiated by NF-κB, its expression product and application
  • A tumor cell-specific effector gene expression vector initiated by NF-κB, its expression product and application
  • A tumor cell-specific effector gene expression vector initiated by NF-κB, its expression product and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Expression of NF-κB RelA in Different Cells

[0052] experimental method:

[0053] Cell culture: HEK-293T (human fetal kidney cells), HepG2 (human liver cancer cells), A549 (human lung cancer cells), HT-29 (human colon cancer cells), HeLa (human cervical cancer cells), SKOV3 (human ovarian cancer cells) cells), PANC-1 (pancreatic cancer cells), MDA-MB-453 (human breast cancer), Hepa1-6 (mouse liver cancer cells), mouse macrophages (RAW264.7), mouse melanoma cells ( B16F10), HL7702 (human normal liver cells) and MRC5 (human embryonic fibroblasts) cell culture, the cells used in the examples were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cell culture uses DEME (Hepa1-6, HEK-293T, HepG2, HeLa, PANC-1, MDA-MB-453, RAW264.7, B16F10, MRC-5) or RPMI 1640 medium (A549, HT-29, SKOV -3, HL7702), 10% (v / v) fetal bovine serum (HyClone), 100units / mL penicillin and 100μg / mL streptomycin culture; culture environment is containing 5% (v / ...

Embodiment 2

[0058] DMP-Display-SBP (cell surface expression of effector genes)

[0059] experimental method:

[0060] Vector construction: construct an expression vector DMP-Display-SBP; the vector contains the DMP sequence and the coding sequence of streptavidin-binding peptide (SBP) that can be expressed in cells. Wherein DMP contains NF-κB response sequence SEQ ID NO.2: (5'-GGG AATTTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3') and minimal promoter sequence SEQ ID NO.3: (5'-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3'). SBP coding sequence SEQ ID NO.4: ATG GAC GAG AAG ACC ACC GGG TGG CGG GGC GGC CAC GTT GTG GAGGGT CTC GCT GGC GAG CTG GAG CAG CTC AGG GCC CGC TTG GAG CAC CAT CCC CAG GGGCAA CGC GAG CCT ATC GAT TAA. The backbone sequence for display expression was cloned from the vector pDisplay TM (Invitrogen); pDisplay TM The protein or polypeptide to be displayed on the cell membrane surface is fused to the N-terminal of the rat Igκ-chain leader sequence (Igκ-chai...

Embodiment 3

[0070] AAV-HBsAg, AAV-SBP, AAV-CRT immunotherapy for mouse tumors

[0071] experimental method:

[0072] Construction of pDMP-Display vector: construct pDMP-Display-SBP vector as in Example 2. The pDMP-Display-HBsAg and pDMP-Display-CRT vectors were constructed and prepared according to the construction procedure of the pDMP-Display-SBP vector.

[0073] The coding sequence of HBsAg is shown in SEQ ID NO.5, and the coding sequence of CRT is shown in SEQ ID NO.6.

[0074] Construction of rAAV-DMP virus vector: AAV-Helper-Free System (Stratagene) was used for experiments. First, three vectors pDMP-Display-SBP, pDMP-Display-HBsAg and pDMP-Display-CRT were constructed. Primers for CRT and HBsAg genes were designed. Using human genome DNA and hepatitis B virus genome DNA as templates, CRT and HBsAg gene sequences were obtained by PCR amplification, respectively. The CMV promoter in the pAAV-MCS vector was replaced with the DMP promoter to construct a vector named pAAV-DMP.

[...

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Abstract

Disclosed are a NF-κB-initiating tumor cell specific-effect gene expression vector and an expression product and an application thereof. The gene expression vector contains two sequence elements, i.e., a promoter sequence for regulating gene expression and an effect gene coding sequence at the downstream of the promoter sequence. The promoter sequence is formed by an NF-κB response sequence and a minimum promoter sequence. When the gene expression vector is imported into a tumor cell, a sequence-specific transcription factor NF-κB in the cell can activate the vector to express an effect gene on the vector. The effect gene expression product is a polypeptide or protein of on the cell surface. The polypeptide or protein on the cell surface not only can be used as a new antigen substance for exciting an in-vivo immune system to attack the cancer cell and developing the efficacy of tumor immunity treatment, but also can be used as a cancer cell artificial marker for tumor imaging, diagnosis, cell separation and the like.

Description

technical field [0001] The invention belongs to the technical field of tumor immunotherapy, and specifically relates to a tumor cell-specific effect gene expression vector activated by transcription factor NF-κB, an expression product and application thereof. Background technique [0002] Human beings have the most urgent expectation for cancer treatment, and the scientific and medical communities have made unremitting efforts to explore it. Cancer is a major disease that plagues human health and threatens human life. Although a lot of basic research on pathogenesis and extensive clinical practice of various treatment methods, so far, the scientific and medical circles have not found a technology that can cure cancer. Although traditional surgical resection plus radiotherapy and chemotherapy have been widely used in cancer treatment, and the latest tumor immunotherapy has also been explored for cancer treatment, the effect of treatment is still far from the patient's expect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63A61K39/00A61P35/00
CPCA61K39/00A61P35/00C12N15/63C12N2830/008A61K39/0011A61K39/001152A61K39/12C12N2710/10343
Inventor 王进科王丹阳戴薇
Owner SOUTHEAST UNIV
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