Non-integration lentiviral vector system and preparation and application thereof

A lentiviral vector, non-integrating technology, applied in the lentiviral vector system and its preparation and application fields, can solve the problem of no manufacturer providing non-integrating lentiviral vectors, and achieve the effects of long-term gene expression and efficient gene transfer.

Active Publication Date: 2011-04-20
SHANGHAI JI KAI GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there are no manufacturers at home and abroad that provide non-integrating lentiviral vectors

Method used

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  • Non-integration lentiviral vector system and preparation and application thereof
  • Non-integration lentiviral vector system and preparation and application thereof
  • Non-integration lentiviral vector system and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] 1 Preparation of carrier

[0069] 1.1 Experimental process description

[0070] From the plasmid pCMV-dR8.2dvpr plasmid template containing the HIV integrase gene, the target gene was caught by PCR method, and the target gene and the target vector were digested respectively. Purify the digested product and carry out directional ligation. The product is transformed into bacterial competent cells. The grown clones are first identified by PCR. The upstream and downstream primers are respectively designed on the carrier and the target gene. The positive clones identified by PCR prove the target gene. Already directional ligated into the destination vector. The positive clones identified by PCR were then sequenced, analyzed and compared, and the correct comparison was the successful construction of fusion protein expression plasmid vectors. The constructed fusion protein expression vector was subjected to ultrapure endotoxin-free extraction.

[0071] 1.2 Experimental flow...

Embodiment 2 Embodiment 1

[0256] Example 1, comparison of GFP conventional lentivirus and GFP non-integrated lentivirus:

example 1

[0257] 1. Cloning the GFP gene into the pLVTH vector:

[0258] Steps: Obtain the target gene GFP by PCR method, digest the PCR product and pLVTH vector, and ligate the digested product with DNA ligase to obtain a recombinant plasmid; transform the recombinant plasmid into competent cells, and screen positive clones by PCR For the cell line, further identify whether the inserted sequence in the positive clone is correct by sequencing; for the correct positive clone, expand the culture, extract the plasmid, and obtain the pGC GFP LV recombinant plasmid DNA.

[0259] 2. Obtain GFP non-integrating lentivirus (mutant GFP LV) according to the method of steps 3-5 in Example 1.

[0260] 3. According to the method of steps 3-5 in Example 1, the only difference is that the pCMV-dR8.2dvpr vector is used instead of the pHelper 1.0 vector to obtain a conventional GFP lentivirus (wild type GFP LV).

[0261] 4. Infect 293T cells with the GFP non-integrating lentiviruses prepared in steps 2 ...

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Abstract

The invention discloses a non-integration slow virus vector system and the preparation and the application thereof. The non-integration slow virus vector system comprises a pLVTH vector, a pHelper1.0 vector, a pCMV-VSV-G vector and host cells. The pLVTH vector, the pHelper1.0 vector and the pCMV-VSV-G vector in the vector system are commonly transfected with the host cells to obtain the non-integration slow virus vector in the host cells by packing. The non-integration slow virus vector system of the invention can provide highly efficient gene transfer, longtime gene expression and wide host cells, cannot randomly integrate carried genes into host gene groups and provides a new method for the gene therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lentiviral vector system and its preparation and application. Background technique [0002] Lentivirus, as a special retrovirus, compared with the commonly used retrovirus vectors and adenovirus vectors, has the ability to infect dividing cells and non-dividing cells, the capacity of the transferred gene fragment is large, the expression time of the target gene is long, and it is not easy to induce The host immune response and other advantages have become the focus of current gene therapy and vector research in transgenic animals, and have been applied to gene therapy research for various diseases. [0003] A typical lentiviral vector system is the HIV-1 vector system, which consists of two parts, namely packaging components and carrier components. The packaging component is constructed by removing the cis-acting sequences required for packaging, reverse transcription and integrat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12R1/93
Inventor 曹跃琼高博逄淑召郑静
Owner SHANGHAI JI KAI GENE TECH CO LTD
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