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PD-1 gene recombinant virus plasmid, construction thereof, recombinant retrovirus Lenti-PD-1-Puro and packaging and application of recombinant retrovirus Lenti-PD-1-Puro

A cas9-pd-1-puro and retrovirus technology, applied in the direction of retroRNA virus, application, virus, etc., can solve the problems of complex antibody preparation and purification process, expensive antibody drugs, high manufacturing cost, etc., and achieve specificity Strong, low cost, easy to culture and expand the effect

Active Publication Date: 2016-06-15
安徽柯顿生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no PD-1 antibody in China, and PD-1 antibody therapeutic drugs are still under development and have not yet entered clinical trials
In addition, the preparation and purification process of PD-1 antibody is complicated, the cycle is long, and the manufacturing cost is high, resulting in expensive antibody drugs. In fact, it is difficult for ordinary people to afford such high treatment costs

Method used

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  • PD-1 gene recombinant virus plasmid, construction thereof, recombinant retrovirus Lenti-PD-1-Puro and packaging and application of recombinant retrovirus Lenti-PD-1-Puro
  • PD-1 gene recombinant virus plasmid, construction thereof, recombinant retrovirus Lenti-PD-1-Puro and packaging and application of recombinant retrovirus Lenti-PD-1-Puro
  • PD-1 gene recombinant virus plasmid, construction thereof, recombinant retrovirus Lenti-PD-1-Puro and packaging and application of recombinant retrovirus Lenti-PD-1-Puro

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 (preparation of PD-1 gene recombinant virus)

[0036] 1) Dephosphorylate the Lenti-CRISPR / Cas9 plasmid (Addgene52961) with EcoR1, Age1 endonuclease and phosphatase at 37°C for 30 minutes to obtain the Lenti-CRISPR / Cas9-Puro plasmid;

[0037] 2) Using a total of 20 base sequences at positions 2859-2878 in the PD-1 gene sequence as the PD-1-specific guide RNA, that is, PD-1gRNA, under the action of T4 ligase (NEBM2200S), the PD-1gRNA primer sequence The complementary strand was incubated at 37°C for 30 minutes, incubated at 95°C for 5 minutes, and then annealed at a rate of 5°C per minute to 25°C to synthesize PD-1 double-stranded DNA;

[0038] 3) Use fast nucleic acid ligase T4 ligase (NEBM2200S) to connect the PD-1 double-stranded DNA to the Lenti-CRISPR / Cas9-Puro plasmid obtained in step 1), and incubate at room temperature for 10 minutes to obtain the recombinant viral plasmid Lenti-CRISPR / Cas9-Puro Cas9-PD-1-Puro;

Embodiment 2

[0039] Embodiment 2 (packaging of recombinant retrovirus Lenti-PD-1-Puro)

[0040] 1) Transfer the recombinant virus plasmid Lenti-CRISPR / Cas9-PD-1-Puro into Stbl3 bacteria, screen with ampicillin, amplify, purify, and sequence, as follows:

[0041] Screening: place the bacterial species transferred into the plasmid on an agar plate containing ampicillin (100 μg / ml), incubate at 37° C. for 12 hours and grow 10 to 20 colonies, and select 3 to 5 colonies to amplify;

[0042] Amplification: Put the above-mentioned selected colonies into 300 ml of LB bacterial culture solution (containing ampicillin, 100 μg / ml), and incubate on a shaker at 37°C for 16 hours, and the bacteria will be amplified in large quantities;

[0043] Purification: Purify with the plasmid extraction kit (Cat. No. 12162) from Qiagen, USA, to obtain 1 to 2 mg of viral plasmid;

[0044] Sequencing: The proposed plasmid was sent to a sequencing company (Laragen, USA) for sequencing, and the viral plasmid with 100...

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Abstract

The invention discloses a PD-1 gene recombinant virus. The collection number of the recombinant virus Lenti-PD-1-Puro is CCTCC No:V201601. According to the PD-1 gene recombinant virus, PD-1gRNA sequences are cloned into a retrovirus plasmid Lenti-CRISPR / Cas9-Puro, and a PD-1 recombinant virus plasmid Lenti-CRISPR / Cas9-PD-1-Puro is obtained; then the PD-1 recombinant virus plasmid Lenti-CRISPR / Cas9-PD-1-Puro, a plasmid pSPAX and pMD2.G are jointly transfected by 293T cells, and packaging of the recombinant virus Lenti-PD-1-Puro is completed. After T cells of peripheral blood of tumor patients are infected by the PD-1 recombinant virus, PD-1 on the T cells is successfully knocked out, the inhibitory state of the T cells of the tumor patients is relieved, the capacity of attacking tumor cells of the T cells embellished by the PD-1 recombinant virus is recovered accordingly, and the effect of immune cell treating is achieved.

Description

technical field [0001] The present invention relates to the field of technical engineering of biotechnology and cell therapy, in particular to a PD-1 gene recombinant virus plasmid, named as virus plasmid KGEN-0626, which was preserved in the Chinese typical Culture Collection Center, the preservation number is CCTCCNO: V201601, the present invention also relates to the packaging of recombinant retrovirus Lenti-PD-1-Puro and the application of the recombinant virus in immunotherapy of tumor cells. Background technique [0002] Cancer has overtaken heart disease as the leading cause of death worldwide. Cancer treatment has made gratifying progress over the past few decades. In addition to surgery, tumor treatment also includes chemotherapy, radiotherapy and targeted drug therapy. Although these methods control the development of cancer to a certain extent, they lack specificity and will kill normal cells while killing tumor cells. In addition, these drugs are highly toxic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01C12N15/12A61K35/768A61P35/00
CPCA61K35/768C07K14/705C12N7/00C12N2740/15032
Inventor 孙昌秀
Owner 安徽柯顿生物科技有限公司
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