Universal type lentivirus vector and preparation method thereof

A lentiviral vector, a general-purpose technology, applied in the field of lentiviral vector and its preparation, can solve the problems of high cost, time-consuming, non-universal lentiviral vector, etc., and achieve the effect of convenient application, rapid screening method and stable expression

Active Publication Date: 2016-10-12
天晴干细胞股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims to solve the problems of cumbersome and time-consuming vector construction steps in the current CAR-T preparation pr...

Method used

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  • Universal type lentivirus vector and preparation method thereof
  • Universal type lentivirus vector and preparation method thereof
  • Universal type lentivirus vector and preparation method thereof

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specific Embodiment approach 1

[0050] Specific embodiment 1: In this embodiment, the universal lentiviral vector uses the pCDH-CMV-MCS-EF1-Puro plasmid as the backbone, and inserts the following elements into the multiple cloning site MCS of the plasmid backbone: CD8αLeader DNA sequence, CD8α hinge region, CD28 Transmembrane region-intracellular region, CD137 intracellular region and CD3ζ; wherein the CD8αLeader DNA sequence is shown in SEQ ID NO: 1 in the sequence listing, the CD8α hinge region is shown in SEQ ID NO: 2 in the sequence listing, and the CD28 transmembrane region - the intracellular region is shown in SEQ ID NO: 3 in the sequence listing, the CD137 intracellular region is shown in SEQ ID NO: 4 in the sequence listing, and the CD3ζ is shown in SEQ ID NO: 5 in the sequence listing.

[0051] In this embodiment, a signal sequence, an extracellular hinge region, a membrane binding region, and an intracellular signal transduction region are constructed. The intracellular signal transduction region c...

specific Embodiment approach 2

[0052] Specific embodiment two: the preparation method of the universal lentiviral vector in this embodiment, the specific steps are as follows:

[0053] 1. Primer design:

[0054] Design the upstream and downstream primers for CD8α hinge region, CD28 transmembrane region-intracellular region, CD137 intracellular region and CD3ζ, respectively named: CD8αF / CD8αR, CD28F / CD28R, CD137F / CD137R, CD3ζF / CD3ζR;

[0055] 2. Acquisition and activation of peripheral blood mononuclear cells:

[0056] Collect adult peripheral blood, extract peripheral blood mononuclear cells, resuspend peripheral blood mononuclear cells (PBMC) with RPMI1640 medium containing 10% FBS and 1000IU / ml IL-2 and dilute to 1×10 6 / ml-1.5×10 6 / ml, take 2ml of the diluent and add it to the 6-well plate coated with the coating solution in advance, and stimulate for 20-28h.

[0057] 3. mRNA extraction and RT reaction:

[0058] Extracting the RNA of the PBMC activated in step 2, and reverse-transcribing it into cDN...

specific Embodiment approach 3

[0065] Specific embodiment 3: The difference between this embodiment and specific embodiment 2 is that the sequence of primer CD8αF in step 1 is: 5'-CCG GAATTC ACCACGACGCCAGCGCCGC-3', primer CD8αF sequence is: 5'- ACCAGCACCCAAAA ATCACAGGCGAAGTCCAGCCCCCT-3'. Others are the same as in the second embodiment.

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Abstract

The invention discloses a universal type lentivirus vector and a preparation method thereof, relates to a lentivirus vector and a preparation method thereof, and aims to solve the problems in a current CAR (Chimeric Antigen Receptor)-T preparation process that the vector construction steps are troublesome, the consumed time is too long, the lentivirus vector cannot be universally used, and the cost is high. The universal type lentivirus vector is characterized by taking pCDH-CMV-MCS-EF1-Puro plasmids as a framework and inserting the following elements of a CD8 (Cluster of Differentiation 8)alpha Leader DNA (Deoxyribose Nucleic Acid) sequence, a CD8alpha hinge region, a CD28 transmembrane region-intracellular region, a CD137 intracellular region and a CD3zeta in MCS (Multiple Cloning Sites) of the plasmid framework. The preparation method comprises the following steps: (1) designing a primer; (2) obtaining and activating a PBMNC (Peripheral Blood Mononulcear Cell); (3) extracting mRNA (Messenger Ribonucleic Acid) and carrying out an RT (Reverse Transcriptase) reaction; (4) preparing recombinant plasmids; (5) compounding a single-chain CD8alpha Leader DNA sequence; (6) connecting a linear vector, a target gene fragment and a CD8alpha Leader fragment, thus obtaining the universal type lentivirus vector. The preparation method disclosed by the invention is used for preparing the lentivirus vector.

Description

technical field [0001] The invention relates to a lentiviral vector and a preparation method thereof. Background technique [0002] T lymphocytes are the natural enemies of tumor cells, play a major role in the tumor immune response, and have a strong killing effect on tumor cells. However, when endogenous T cells are used for tumor immunotherapy, the target antigen needs to be processed before it can interact with the major histocompatibility complex (MHC) on the surface of the target cell, which is what we often call "MHC Restriction". However, the process of tumor immunoediting will reduce the expression of MHC on the surface of tumor cells, destroy the antigen processing process, and reduce the immunogenicity of peptides. Such a long-term immune escape mechanism can enable tumor cells to successfully evade the attack of T cells, and the tumor proliferates rapidly. [0003] Adoptive cellular immunotherapy (ACI) is currently one of the more effective treatments for mali...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66
CPCC12N15/66C12N15/86C12N2740/15043C12N2800/107
Inventor 刘春香张怡芦慧颖石佳宁刘艳青
Owner 天晴干细胞股份有限公司
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