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MSGV recombinant carrier as well as preparation method and application thereof

A technology for recombining vectors and vectors, which is applied in the field of genetic engineering and can solve the problems of unsatisfactory safety mismatch rate and other problems

Inactive Publication Date: 2018-05-29
HENAN HUALONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are unsatisfactory in improving security and reducing the mismatch rate.

Method used

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  • MSGV recombinant carrier as well as preparation method and application thereof
  • MSGV recombinant carrier as well as preparation method and application thereof
  • MSGV recombinant carrier as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Construction of pMSGV-GDZ recombinant vector

[0072] (1) Design and chemically synthesize the TRGC-CD3ζ-P2A-TRDC-CD3ζ gene fragment shown in SEQ ID NO.5;

[0073] (2) The MSGV vector was double-enzyme-digested with PacI and XhoI. The enzyme-digestion system is shown in Table 1, and the MSGV vector was gel-recovered after digestion;

[0074] Table 1 enzyme digestion system

[0075] Reagent

Dosage

PacI (10U / μL)

2μL

XhoI (10U / μL)

2μL

10×H buffer

2μL

pMSGV-GDZ vector

1μg

h 2 o

Supplement to 20 μL

[0076] (3) Using T4 DNA ligase, carry out DNA ligation reaction between the synthesized SEQ ID NO.5 sequence and the MSGV vector recovered from the gel. The ligation system is shown in Table 2. The ligation reaction was kept at 22°C for 30min, and then at 70°C Inactivate for 5 minutes;

[0077] Table 2 Connection system

[0078] Reagent

Dosage

MSGV restriction fragment

...

Embodiment 2

[0082] Example 2 A large amount of extraction of pMSGV-GDZ recombinant vector

[0083] Take out the positive bacteria expressing the pMSGV-GDZ recombinant vector from -80°C, culture overnight after streaking on the plate, pick 2-4 single clones the next day, and shake the bacteria in a small volume (5mL) for 6-8h; The ratio was 1:100 and shaken, and then the plasmid was extracted with a large amount of plasmid extraction kit (Omega Endo-free Plasmid Maxi Kit, Cat. No. D6926-03). Plasmid extraction involves the following steps:

[0084] (1) Take an appropriate amount of bacterial solution and place it in a new 50mL centrifuge tube, centrifuge at 4900g for 10min at room temperature, discard the supernatant and continue to add an appropriate amount of bacterial solution until all the bacterial solution is centrifuged to obtain a precipitate;

[0085] (2) Add 10mL of buffer A to the bacterial pellet after centrifugation, and resuspend on a shaker;

[0086] (3) Add 10mL of buffer...

Embodiment 3

[0098] Example 3 Construction of pMSGV-GDZ-NY retrovirus expression vector

[0099](1) Using the Golden Gate molecular cloning technology, the synthetic NY-ESO-1-specific TCR double-strand V region DNA sequence (SEQ ID NO.7, SEQ ID NO.8) was connected to pMSGV-GDZ In the recombinant vector, the connection system is shown in Table 3;

[0100] Table 3 connection system

[0101] Reagent

Dosage

pMSGV-GDZ recombinant vector

100ng

SEQ ID NO.7

50ng

SEQ ID NO.8

50ng

BbsI (5U / μL)

2μL

T4 DNA ligase (350U / μL)

1μL

10x T4 DNA Ligase Buffer

2μL

h 2 o

Supplement to 20 μL

[0102] (2) The ligation reaction conditions are: 37°C / 10min, 16°C / 15min, a total of 5 cycles; 80°C / 5min, a total of 80min; after adding 1 μL of ATP buffer and 1 μL of exonuclease (Plasmid Safe Exonuclease), 37°C / 60min, 70°C / 30min, digest linear DNA to obtain Figure 4-5 The indicated pMSGV-GDZ-NY retroviral expression vector...

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Abstract

The invention provides an MSGV recombinant carrier as well as a preparation method and application thereof. The recombinant carrier comprises a gene segment comprising a TRGC gene, a TRDC gene and a CD3-zeta gene. A C area of alpha-beta TCR on the MSGV carrier is replaced by an extracellular C area of gamma-zeta TCR as well as a transmembrane area and an intracellular area of the CD3-zeta, so thatthe mismatch rate of exogenous TCR molecules and endogenous TCR is obviously reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a MSGV recombinant vector and its preparation method and application. Background technique [0002] In recent years, breakthroughs have been made in adoptive immunotherapy using genetically engineered T cells (such as CAR-T, TCR-T). Remission (S.L.Maude et al.Chimeric antigen receptor T cells for sustainedremissions in leukemia.Engl.J.Med.,371,1507-1517(2014)). However, T cell-based immunotherapy faces safety issues such as off-target effects and cytokine release syndrome. The reason for TCR-T off-target is that on the one hand, the selected TCR molecule can recognize the antigenic peptide of the non-target protein, and on the other hand, it is due to the mismatch between the exogenous TCR molecule and the endogenous TCR molecule to produce a hybrid TCR, which leads to transplantation. The occurrence of animal-versus-host disease (GvHD). [0003] At present, research...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/12C12N7/01A61K48/00A61K35/17A61P35/00
CPCA61K35/17A61K48/005C07K14/7051C12N7/00C12N15/86C12N2740/15021C12N2740/15043C12N2740/15052C12N2800/107
Inventor 韦丹马亚锋连杰秦志华范宇清皇甫晶晶
Owner HENAN HUALONG BIOLOGICAL TECH
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