High-activity T-cell promoter and application thereof

A promoter and cell technology, applied in the field of molecular biology, can solve problems such as difficulty in antibody expression reaching therapeutic concentrations, lack of promoters, etc.

Active Publication Date: 2015-07-01
SHANGHAI CELL THERAPY RES INST +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although in previous studies, we have established an adenovirus-mediated full-length antibody gene expression system and initially realized the expression of a functionally active full-length antibody in T cells, due to the lack of a suitable promoter, the expression level of the antibody Difficult to achieve therapeutic concentrations

Method used

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  • High-activity T-cell promoter and application thereof
  • High-activity T-cell promoter and application thereof
  • High-activity T-cell promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1: Construction of a dual-luciferase detection system with commonly used strong promoters

[0108] 1. According to the coding sequence of the firefly luciferase (Firefly luciferase, FLuc for short) gene in the psi-CHECK2 plasmid (purchased from Promega), design a pair of PCR-specific amplification primers (upstream primer F1, add EcoRI restriction Restriction site and protection base; downstream primer R1, plus SalI restriction site and protection base). The sequence of F1 and R1 is as follows:

[0109] F1: GCCgaattcGCCACCATGGAAGACGCC (SEQ ID NO: 7), wherein lowercase letters represent the EcoRI restriction site;

[0110] R1: TGTgtcgacTTACACGGCGATCTTTCCGC (SEQ ID NO: 8), wherein the lowercase letters represent the SalI restriction site.

[0111] Using the psi-CHECK2 plasmid as a template, amplify the FLuc gene coding sequence, digest it with EcoRI+SalI double enzymes, and load it into pENTR that has also been digested with EcoRI+SalI double enzymes TM Vecto...

Embodiment 2

[0129] Example 2: Activity detection of commonly used strong promoters in T cell lines

[0130] The low-passage Jurkat T and K562 cell lines (both purchased from ATCC) in good growth state were divided into 1 × 10 4 Cells / well spread 96-well plate, set at 37°C, 5% CO 2 Culture in the incubator for 24 hours; according to MOI=5, respectively infect Ad35-CMV-2Luc, Ad35-CMVi-2Luc, Ad35-EF1α-2Luc, Ad35-EF1αi-2Luc, Ad35-CAG-2Luc, Ad35-CCAU- 2Luc and other 6 kinds of recombinant viruses were set up with 4 duplicate wells for each group; 2 Incubator culture; after 24 hours, the cells were lysed, and placed in a microplate reader to measure the enzymatic activity of RLuc by a dual-luciferase detection kit (purchased from Promega Company), and the enzymatic activity of FLuc was used as an internal reference to obtain RLuc / FLuc relative ratio. The specific operation steps were completed according to the instructions of the kit.

[0131] The results show that (see Figure 2A-2B ), ...

Embodiment 3

[0132] Example 3: T cell strong promoter construction based on EF1α promoter

[0133] According to the nucleotide sequences of CCEF promoter, TEF promoter, TCEF promoter, CCEFi promoter, TEFi promoter, TCEFi promoter shown in SEQ ID NO: 1-SEQ ID NO: 6 (promoter pattern diagram sees image 3 ), and introduced XbaI at its upstream, and introduced the enzyme cutting site EcoRI at its downstream, entrusted Shanghai Jierui Biology Co. CMV promoter in the Rluc vector), named respectively as pDC315-CCEF-RLuc, pDC315-TEF-RLuc, pDC315-TCEF-RLuc, pDC315-CCEFi-RLuc, pDC315-TEFi-Rluc, pDC315-TCEFi-Rluc (vector map See Figure 4 ). After transforming each of the above plasmids into DH5α bacteria, Shanghai Sangong was commissioned to determine the DNA sequence of each promoter, and it was found that the sequence of each promoter remained stable.

[0134] Co-transfect pDC315-CCEF-RLuc, pDC315-TEF-RLuc, pDC315-TCEF-RLuc, pDC315-CCEFi-RLuc, pDC315-TEFi-Rluc, pDC315-TCEFi-Rluc and pPE35-Fl...

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Abstract

The invention belongs to the field of molecular biology and relates to a high-activity T-cell promoter and an application thereof. Particularly, the high-activity T-cell promoter comprises an element 1 and an element 2, wherein the element 1 is an EF1 alpha promoter and/or an EF1 alpha promoter containing an intron; and the element 2 is any one or more of an mCMV promoter, an hCMV promoter and a CD3e promoter. The high-activity T-cell promoter provided by the invention can be used for efficiently expressing a foreign gene in a T cell, has a stable sequence and is free from sequence loss during the transfer process of a prokaryotic cell and a eukaryotic cell. The high-activity T-cell promoter is applicable to driving the high-efficiency expression of the foreign gene, especially a full-length antibody gene, in the T cell during the process of adoptive cellular immunotherapy.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a promoter, in particular to an artificially synthesized chimeric promoter, which has high activity in T cells. The present invention also relates to a recombinant vector containing the promoter, a recombinant virus, and the use of the promoter to control exogenous genes in transgenic T cell therapy. Background technique [0002] Immunotherapy against malignant tumors has developed rapidly in recent years and has achieved remarkable clinical efficacy. In 2011, Nature and JCO, the top journal of clinical oncology, respectively published review articles with the same title "The era of tumor immunotherapy has come" (Nature.2011; 480(7378):480; J Clin Oncol.2011; 29(36): 4828), believed that tumor immune cell therapy is about to usher in a new round of research climax, and may occupy a very important position in tumor treatment in the future. In the field of industrialization, accordi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/13C12N15/85C12N15/86C12N5/10A61K48/00A61K39/395A61P35/00
Inventor 钱其军金华君吕赛群刘品一李振海李林芳黎江吴红平吴孟超
Owner SHANGHAI CELL THERAPY RES INST
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