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Attached type lentiviral vector as well as preparation method and application thereof

A technology of lentiviral vectors and vectors, applied in the biological field, to achieve the effect of broad market prospects

Active Publication Date: 2015-11-11
河南普诺易生物制品研究院有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the presence of MAR sequences for mediating lentiviral vector attachment

Method used

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  • Attached type lentiviral vector as well as preparation method and application thereof
  • Attached type lentiviral vector as well as preparation method and application thereof
  • Attached type lentiviral vector as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Construction of attached lentiviral vectors:

[0053] 1. Construction of pLVX-IRES-ZsGreen2 vector

[0054] (1) Artificially synthesized sequence A

[0055] According to the pLVX-IRES-ZsGreen1 sequence (Cat.Nos.632187), design and synthesize the A sequence (as shown in SEQ ID NO.1, synthesized by General Biosystems (Anhui) Co., Ltd.), and a PacI restriction site is introduced into the sequence A ( TTAATTTA ) and XmaI restriction site ( CCCGGG ).

[0056] (2) Construction of pLVX-IRES-ZsGreen2 vector

[0057] The synthetic sequence A and pLVX-IRES-ZsGreen1 plasmids were digested with BsrGI and Bsu36I. Use conventional enzyme digestion methods and agarose gel electrophoresis to identify the enzyme digestion results, and recover the A-sequence DNA fragments and vectors from the gel and connect them.

[0058] The enzymatic digestion system of Sequence A is shown in Table 1 below. After mixing well, place in a water bath at 37°C for 6 hours.

[0059] Table 1 Sequenc...

Embodiment 2

[0126] Application of attached lentiviral vector:

[0127] (1) Cloning of miR-145

[0128] PCR primers P3 and P4 were designed according to the human miR-145 precursor sequence (Genbank accession number GQ292874.1, shown in SEQ ID NO.6), and the primer sequences were as follows:

[0129] P3: 5'-CCG CTCGAG GAATGGTTGGTTTAAATCCACCC-3' (as shown in SEQ ID NO.7),

[0130] P4: 5'-CCG GGATCC CATGCCTGATGGTGTCCTG-3' (as shown in SEQ ID NO.8),

[0131] The underline is the restriction site, wherein P3 contains the XhoI restriction site, and P4 contains the BamHI restriction site.

[0132]Use primers and extracted human genomic DNA templates for PCR amplification. The amplification reaction system is shown in Table 8 below. The reaction program is: 95°C pre-denaturation for 5 minutes, 94°C for 40s, 50°C for 30s, 72°C for 40s, 30 cycles, Finally, extend at 72°C for 5 min. After the reaction, 10 μL of the reaction product was taken for electrophoresis detection in 1.5% agarose gel,...

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Abstract

The invention discloses an attached type lentiviral vector as well as a preparation method and an application thereof and belongs to the biotechnology field. The attached type lentiviral vector is obtained through co-transfection of host cells on a pLRZM vector and a packaging system, the pLRZM vector is obtained by replacing a woodchuck hepatitis virus post-transcriptional control element in a pLVX-IRES-ZsGreen1 with a human-derived MAR sequence or a homologous sequence thereof with the homology of more than 95%, a sequence of the woodchuck hepatitis virus post-transcriptional control element is shown in SEQ ID NO.2, and the human-derived MAR sequence is shown as 14th-2161st basic groups in SEQ ID NO.3. The attached type lentiviral vector is attached on chromosome of the host cell but not being integrated into the chromosome of the host cell, can mediate stable, high-efficiency and durable expression of a foreign gene in mammalian cells, can be applied to gene therapy and has a broad market prospect.

Description

technical field [0001] The invention relates to an attached lentiviral vector, a preparation method and application of the attached lentiviral vector, and belongs to the field of biotechnology. Background technique [0002] With the deepening of the understanding of the molecular mechanism of diseases and the development of biotechnology, gene therapy has become a new way to treat diseases, and the diseases treated have also expanded from genetic diseases to infectious diseases, nervous system diseases and tumors. Currently, the vector systems used for gene therapy can be divided into two categories: viral vectors and non-viral vectors. Lentiviral vector is a special kind of retrovirus. Compared with commonly used retroviral vectors and adenoviral vectors, it can not only infect dividing cells, but also infect non-dividing cells (such as nerve cells, hematopoietic stem cells, muscle fiber cells, etc.) and liver cells, etc.), and has the advantages of accommodating large fra...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66
Inventor 王天云贾岩龙张俊河赵春澎李琴王小引
Owner 河南普诺易生物制品研究院有限公司
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