Multifunctional immune killing transgenic cell as well as preparation method and use thereof

A technology of transgenic cells and killing cells, which is applied in gene therapy, cells modified by introducing foreign genetic material, antibacterial drugs, etc., can solve the problems of low transfection rate and no antibody gene, and achieve the effect of inhibiting proliferation

Active Publication Date: 2011-10-19
SHANGHAI CELL THERAPY GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although there have been reports (CN1732267, CN1629285, and CN1705739) on transfecting exogenous genes into T cells, the gene transfection vector system commonly used at present has a low transfection rate for cellular immune effector cells with cytotoxicity, and it is difficult to make exogenous The gene is expressed at a high level in its cells, and so far, there has not been any report on the expression of antibody genes containing human constant regions in cellular immune effector cells
Therefore, before this, there are no reports of transgenic cells with cytotoxicity and high-level expression of antibodies containing human constant regions

Method used

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  • Multifunctional immune killing transgenic cell as well as preparation method and use thereof
  • Multifunctional immune killing transgenic cell as well as preparation method and use thereof
  • Multifunctional immune killing transgenic cell as well as preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Construction of Adenovirus Shuttle Plasmids pDC339 and pDC359 and AAV ITR-Containing Adenovirus Shuttle Plasmids pDC639 and pDC659

[0048] 1. Construction of pDC339 vector:

[0049] Step 1: Build pDC315-Linker1

[0050] SEQ ID NO: 1:5'-AGCTCGAATTCGTCGACAAGCTTGGATCCCTCGAGGCTAGCACTAGTCATATGC-3'

[0051] SEQ ID NO: 2: 5'-TCGAGCATATGACTAGTGCTAGCCTCGAGGGATCCAAGCTTGTCGACGAATTCG-3'

[0052] Mix 100nmol of SEQ ID NO: 1 and SEQ ID NO: 2 single-stranded DNA together, through high temperature denaturation (95°C for 10 minutes), slow annealing and renaturation (gradually cool to room temperature) to synthesize a double-stranded DNA linker, and end to form EcoRI and XhoI cohesive ends, respectively.

[0053] The vector pDC315 (purchased from Microbix Biosystem Inc. Toronto) was digested with EcoRI and SalI, and the vector fragment was recovered by electrophoresis (see the recovery kit NucleospinExtract II for specific steps), and connected with linker1 to obtain pDC31...

Embodiment 2

[0104] Example 2: Backbone plasmid construction of adenovirus

[0105] 1. Construction of pCLON9 vector

[0106] Synthesis of SEQ ID NO: 8: 5'-AATTGACCGGTCTCGAGACTAGTGGATCCGCGGCCGCATCTAGATTAATTA-3';

[0107] and SEQ ID NO: 9: 5'-AGCTTAATTAATCTAGATGCGGCCGCGGATCCACTAGTCTCGAGACCGGTC-3';

[0108] After denaturation and renaturation, the EcoR I and HindIII sites of the pUC19 vector were inserted to obtain the pCLON9 vector (the method is the same as above) (see Figure 5 ).

[0109] 2. Construction of pPE3 vector

[0110] Synthesize the following primers:

[0111] SEQ ID NO: 10:

[0112] 5'-ggACTAGTTTCGCGCCCTTTCTCAAATTTAAGCGC-3';

[0113] SEQ ID NO: 11: 5'-CCCCGGGTACTCTAGTTAATTAACTATTACTTAGCCAATGTGGAGC-3';

[0114] SEQ ID NO: 12:

[0115] 5'-GCTCCACATTGGCTAAGTAATAGTTAATTAACTAGAGTACCCGGGG-3';

[0116] SEQ ID NO: 13: 5'-cccAAGCTTTTTGGAATTGTTTGAAGCTGTAAAC-3';

[0117] Using the type 5 adenovirus backbone plasmid pBGHE3 (purchased from Microbix Biosystem Inc. Toronto) as a te...

Embodiment 3

[0243] Example 3: Construction of a shuttle plasmid carrying a full-length antibody gene containing a human constant region

[0244] Table 2: Overview of Antibodies AT1-AT7

[0245] Antibody name

[0246] AT6

[0247] SEQ ID NO: 30 (AT1 expression cassette, including light chain, IRES, heavy chain genes):

[0248] (EcoRI)

[0249]ACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTGAGTGGATCCTTCTAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACG...

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Abstract

The invention relates to a multifunctional immune killing transgenic cell and in particular relates to a multifunctional immune killing transgenic cell which has cell killing toxicity and expressing an antibody containing a human constant region. The gene of the antibody containing the human constant region is transfected into a cell with cell killing toxicity through coding, and is expressed in the cell at a high level so as to realize the double effects of cell immunity and antibody and inhibit multiplication of tumor and virus. The invention also relates to a preparation method and use of the multifunctional immune killing transgenic cell.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell biology, and relates to a multipotent immune killing transgenic cell. Specifically, the invention relates to a transgenic immune cell capable of high-level expression of an antibody containing a human constant region and having cell killing toxicity. Because this kind of transgenic immune cells not only has cytotoxicity, that is, cellular immunity, but also can secrete antibodies at a high level, that is, humoral immunity, we named this kind of cells pluripotent immune killer transgenic cells (pluripotent immune killer cells, referred to as PIK) ). The present invention also relates to the preparation method and application of the pluripotent immunokilling transgenic cell. Background technique [0002] "Immunity" can be divided into cellular immunity and humoral immunity. Cellular immunity is the direct effect of cells to remove foreign substances. The participating cells are called cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61K48/00A61P35/00A61P31/12A61P31/04A61P37/02
Inventor 钱其军李琳芳吴红平金华君孙艳丁娜徐凤青吴孟超
Owner SHANGHAI CELL THERAPY GRP CO LTD
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