Mutant encoding gene of human 4IgB7-H3 and application thereof in regulating immunity

A 4igb7-h3, encoding gene technology, applied in the direction of anti-animal/human immunoglobulin, application, genetic engineering, etc., can solve the problem that there is no 4IgB7-H3 mutation encoding gene reported, and achieve the effect of enhanced inhibitory effect

Active Publication Date: 2021-07-23
白素梅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The provision of various mutants and functional studies of 2IgB7-H3 and 4IgB7-H3 also provides a new way for the above-m

Method used

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  • Mutant encoding gene of human 4IgB7-H3 and application thereof in regulating immunity
  • Mutant encoding gene of human 4IgB7-H3 and application thereof in regulating immunity
  • Mutant encoding gene of human 4IgB7-H3 and application thereof in regulating immunity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of plasmid containing 4IgB7-H3 wild-type coding gene and 4IgB7-H3 mutant coding gene

[0046] 1. Materials and methods

[0047] After PCR, the artificially synthesized wild-type 4IgB7-H3 coding gene (SEQ ID NO: 2, NCBI Reference Sequence: NM_001024736.1) and 4IgB7-H3 mutant coding gene (SEQ ID NO: 1) were constructed into PGMLV-PA7 and PGMLV-PA6, and Blasticidin and puro were used as screening markers, respectively. The vector was transformed into competent cells by conventional techniques in the field, and the competent cells were plated and placed upside down in a 37°C constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.) for overnight culture. On the second day, single clones were selected, and two of each carrier clone were selected and sent to the sequencing company for sequencing identification. After comparison, the sequence of the insert fragment in the recombinant clone was completely consistent with the sequenc...

Embodiment 2

[0072] The packaging of embodiment 2 lentivirus and the mensuration of virus titer

[0073]Cell line: HEK 293T, the packaging cell of lentivirus, is an anchorage-dependent epithelioid cell, and the growth medium is DMEM (containing 10% FBS). Adherent cells grow and proliferate in culture to form a monolayer of cells.

[0074] Strain: E. coli strain DH5α. For amplification of lentiviral vectors and helper packaging vector plasmids.

[0075] Lentiviral packaging system: The successfully constructed lentiviral recombinant plasmids and packaging plasmids were extracted using Qiagen's plasmid extraction kit. The obtained plasmid DNA was dissolved in sterile TE, and its concentration and purity were measured by ultraviolet light absorption method to ensure that the A260 / A280 of the extracted plasmid DNA was between 1.8 and 2.0.

[0076] 1. Lentiviral packaging

[0077] 1) HEK 293T cell division: The day before transfection, subculture the grown cells into a 10cm culture dish at ...

Embodiment 3

[0106] Example 3 Comparison of Mutant and Wild-type HEK293T Cell Effects in Vitro

[0107] 1. Lentivirus infection of target cells

[0108] On the first day, target cells HEK293T (purchased from the Shanghai Chinese Academy of Sciences Cell Bank) were inoculated in a culture dish at an appropriate ratio.

[0109] On the next day, before infection, the virus stock solution was taken out from the -80°C refrigerator and melted in an ice bath, and the virus stock solution was diluted with a fresh medium containing 5 μg / ml Polybrene according to an appropriate MOI value, and the original culture medium of the control group and the treatment group was aspirated. Base, the dilution containing the lentivirus negative control (NC-) was added to the cells of the control group, and the dilution containing the lentivirus solution was added to the cells of the treatment group.

[0110] On the third day, the culture medium was replaced, and the culture medium containing the lentivirus was ...

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Abstract

The invention discloses a new mutant encoding gene of human 4IgB7-H3 and application thereof in regulating immunity. The nucleotide sequence thereof is shown in SEQ ID NO:1. The mutant encoding gene of human 4IgB7-H3 has an enhanced biological function as compared to the wild-type gene, that is, the binding of the mutant encoding 4IgB7-H3 with an inhibitory receptor has an enhanced inhibitory effect on the proliferation and activation of T cells. 4IgB7-H3, as a costimulatory molecule, plays an important role in the processes of T and B cell activation, helper T cell differentiation, signal transduction and cytokine secretion of effector cells. The mutant encoding gene of human 4IgB7-H3 plays a role in solving and treating the main reason why it is difficult to induce tumor specific T cell activation in tumor escape from immune surveillance, and has prominent and wide application prospects in preparation of genomic engineering, gene recombination, immunotherapeutic drugs, vaccines and protein substitute therapies for treating malignant tumors and infectious diseases based on T cells and dendritic cells, allergy relief and the like.

Description

technical field [0001] The invention relates to the field of biomedical immunotherapy, in particular to a human 4IgB7-H3 mutation coding gene and its application in regulating immunity. Background technique [0002] The dynamic balance achieved by the orderly regulation of positive / negative costimulatory signals (also known as co-stimulatory signals, Co-stimulatory signals) plays an important role in the body's resistance to the invasion of foreign antigens and the occurrence of autoimmune diseases. effect. The activation or proliferation of T lymphocytes (referred to as T cells) depends on positive / negative dual signals, which are necessary for the effective stimulation of T cell antigen specificity and the timely termination of immune effects. The imbalance of immune regulation, regardless of inhibition or excessive activation, is an immune abnormality, which affects the body's immune response and causes various immune diseases, including tumors, inflammation, and autoimm...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/705C12N15/867C12N5/10C12N15/113A61K45/00A61K38/17A61P35/00A61P37/08A61P37/06A61P37/02
CPCC07K14/70532C12N15/86C12N5/0686C12N5/0694C12N15/1138A61K45/00A61P35/00A61P37/08A61P37/06A61P37/02C12N2740/15043C12N2800/107C12N2510/00C12N2310/141A61K38/00A61K38/17C07K14/435C07K14/705C12N5/10C12N15/113C12N15/867C12N15/11A61K39/00C12N5/06C07K16/18
Inventor 白素梅
Owner 白素梅
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