Preparation method of recombinant vectors CAR-CD244-antiPSMA

一种car-cd244-antipsma、CD244的技术，应用在重组载体CAR-CD244-antiPSMA的制备领域，能够解决尚未建立CAR-NK细胞肿瘤免疫治疗应用先例等问题，达到使用范围广、制备成本低的效果

Active Publication Date: 2018-12-28
GUOJIAN CHENGNUO BIOTECHNOLOGY (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, an effective method for the preparation of CAR-NK cells and its application precedent in tumor immunotherapy have not been established

Method used

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  • Preparation method of recombinant vectors CAR-CD244-antiPSMA
  • Preparation method of recombinant vectors CAR-CD244-antiPSMA
  • Preparation method of recombinant vectors CAR-CD244-antiPSMA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Preparation of recombinant vector CAR-CD244-antiCD19 and recombinant vector CAR-CD244-antiPSMA

[0056] 1. The specific antibody-encoding gene is amplified by PCR using primers with BfuAI restriction endonuclease recognition sites at the end;

[0057] The specific antibody coding gene sequences used in this example are the partial sequence of CD19 antibody (antiCD19) (sequence listing sequence 2) and the partial sequence of PSMA antibody (antiPSMA) (sequence listing sequence 3);

[0058] 2. Purify the PCR products of step 1 respectively;

[0059] 3. Carry out single enzyme digestion (BfuAI) to the PCR product purified in step 2, and simultaneously carry out single enzyme digestion (BfuAI) to the cloning vector containing the transformed CD244 backbone;

[0060] 4. Purify the digested product;

[0061] 5. After the purified product is prepared according to the molar ratio of the antibody fragment and the cloning carrier containing the modified CD244 backbone...

Embodiment 2

[0064] Embodiment 2, preparation of recombinant lentiviral vector

[0065] 1. Using the recombinant vector CAR-CD244-antiCD19 prepared in Example 1 and the recombinant vector CAR-CD244-antiPSMA as templates respectively, PCR amplification was performed using primers with BamHI and XbaI restriction endonuclease site sequences;

[0066] 2. Purify, digest (BamHI and Xba I) and purify the PCR products respectively;

[0067] 3. Use T4 ligase to connect the products of step 2 into the Plenti lentiviral vector (named pLentiCMV / TO eGFP Puro (w159-1), purchased from addgene, website: http: / / www.addgene.org / 17481 / ) between BamHI and XbaI sites;

[0068] 4. Transformation, plating, picking single clones to extract plasmids for sequencing, and named the recombinant lentiviral vectors with correct sequencing as recombinant lentiviral vectors Plenti-CAR-CD244-antiCD19 and Plenti-CAR-CD244-antiPSMA respectively.

[0069] The schematic diagram of the recombinant lentiviral vector Plenti-CA...

Embodiment 3

[0070] Example 3, preparation and concentration of lentivirus

[0071] 1. Preparation of lentivirus

[0072] Use the recombinant lentiviral vector Plenti-CAR-CD244-antiCD19 prepared in Example 2 and psPAX2 (purchased from addgene, website at https: / / www.addgene.org / 12260 / ), pMD2.G (purchased from addgene, website 293T cells (about 3×10 6 293T cells were plated on a 10cm dish), and replaced with fresh medium (DMEM medium, added with 10% fetal calf serum) after 8 hours, and the supernatant was collected every 24 hours thereafter and added with fresh medium (DMEM medium, added with 10% Fetal bovine serum), collected 3 times in total to obtain lentiviral supernatant A.

[0073] Using the recombinant lentiviral vector Plenti-CAR-CD244-antiPSMA prepared in Example 2 and the psPAX2, pMD2.G vectors in a mass ratio of 5ug:3.2ug:1.8ug to transfect 293T cells (about 3×10 6 293T cells were plated on a 10cm dish), and replaced with fresh medium (DMEM medium, supplemented with 10% fetal ...

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Abstract

The invention relates to a preparation method of recombinant vectors CAR-CD244-antiPSMA, comprising the following steps: step 1, performing PCR amplification on a specific antibody coding gene by using a primer with a BfuAI restriction endonuclease recognition site at the terminal, wherein the sequence of the specific antibody coding gene is a partial sequence of aPSMA antibody, which is sequence3 of a sequence table; step 2, purifying the PCR product of step 1; step 3, performing single digestion on the PCR product purified in step 2 at the BfuAI restriction endonuclease recognition site, and simultaneously performing single digestion on a cloning vector containing a transformed CD244 skeleton at the BfuAI restriction endonuclease recognition site; step 4, purifying the digested products; and step 5, proportioning the purified products according to a molar ratio 3: 1 of the antibody fragment to the cloning vector containing the transformed CD244 skeleton, ligating with a T4 ligase, transforming, coating, and picking monoclonal extraction plasmids for sequencing; and naming the correctly ligated plasmids as recombinant vectors CAR-CD244-antiPSMA.

Description

[0001] This application is a divisional application, the application number of the original application is 201710209997.5, the application date is March 31, 2017, and the title of the invention is "a CAR-NK cell and its preparation method and application". technical field [0002] The invention relates to the field of biotechnology, in particular to a method for preparing a recombinant vector CAR-CD244-antiPSMA. Background technique [0003] Cellular biotherapy is a new cancer treatment method using the autoimmune system, and it is a treatment mode with significant curative effect. It uses molecular biology technology and cell engineering technology to culture and expand the immune cells collected from the patient's body with biological agents, and then reinfuse them back into the patient's body, so as to stimulate and enhance the body's own immune function and achieve surveillance. And the purpose of killing diseased cells or repairing damaged cells. [0004] Natural kille...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12N15/62A61K35/17A61P35/00
CPCA61K35/17C07K14/70503C07K16/2803C07K16/3069C07K2319/02C07K2319/03C07K2319/33C12N5/0646C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 顾雨春尹乐
Owner GUOJIAN CHENGNUO BIOTECHNOLOGY (BEIJING) CO LTD
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