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Auxiliary plasmid vector for packaging AAV virus as well as construction method and application of auxiliary plasmid vector

A technology of auxiliary plasmid and construction method, which can be applied in the directions of virus/phage, single-stranded DNA virus, and introduction of foreign genetic material using a vector, which can solve the difficulties in the commercial production process of viral vectors, increase the production cost of viral vectors, and increase the production of viral vectors. efficiency and other issues, to achieve the effect of enhancing the effect of eukaryotic expression, improving the titer of packaging virus, and increasing the therapeutic value

Pending Publication Date: 2022-06-03
济南宜明医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the commercial production process of the virus vector is very difficult, and it is difficult to obtain a high production efficiency of the virus vector, which greatly increases the production cost of the virus vector

Method used

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  • Auxiliary plasmid vector for packaging AAV virus as well as construction method and application of auxiliary plasmid vector
  • Auxiliary plasmid vector for packaging AAV virus as well as construction method and application of auxiliary plasmid vector
  • Auxiliary plasmid vector for packaging AAV virus as well as construction method and application of auxiliary plasmid vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of YM-HELPER plasmid

[0036] The construction idea is: design specific PCR primers to amplify the kanamycin sulfate resistance gene and SV40ori sequence fragment (sequence such as SEQ ID NO: 1) contained in the backbone plasmid, wherein the backbone plasmid map is as follows figure 1 As shown, the template plasmid (containing E2A, E4 gene sequence, the template plasmid vector map is as shown in figure 2 shown), after double digestion with Bsp1407I / MssI, the two were ligated to obtain the YM-HELPER plasmid (see image 3 ).

[0037] Specific steps are as follows:

[0038]PCR: Design specific primers required to amplify DNA fragments to amplify the backbone plasmid (plasmid source Shandong Weizhen Biotechnology Co., Ltd.), see Table 1 for details.

[0039] Table 1: PCR primer sequences

[0040]

[0041] The PCR system is shown in Table 2:

[0042] Table 2: PCR System

[0043] reagent volume / μl Template(plasmid) 1 Pr...

Embodiment 2

[0055] Example 2: AAV virus packaging application

[0056] In order to evaluate the packaging application effect of the constructed plasmid, a comparative example is set to compare with the helper plasmid vector of the present invention.

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Abstract

The invention discloses a helper plasmid vector as well as a preparation method and application thereof, the helper plasmid vector contains an SV40 replication initiation site (SV40origin of replication, SV40ori) sequence, and the helper plasmid vector can significantly enhance the eukaryotic expression effect, improve the packaging virus titer and contribute to increasing the treatment value of an rAAV vector.

Description

technical field [0001] The present invention relates to the technical field of gene and cell therapy drug carriers, in particular to a helper plasmid vector used for adeno-associated virus (AAV) packaging, which can increase virus titer, a preparation method and application thereof. Background technique [0002] Adeno-associated virus (AAV) is a single-stranded DNA, non-enveloped, icosahedral virus belonging to the genus Dependent Parvovirus. AAVs are not pathogenic in humans, they were initially found to be contaminants of adenovirus preparations and require co-infection with adenovirus or herpesvirus for efficient replication. AAV virus packaging is currently the most commonly used AAV helper-free system (AAV Helper-Free System), which can produce recombinant adeno-associated virus without helper virus. This system is the co-transfection of AAV production cells and three plasmids. The three plasmids are the shuttle plasmid carrying the target gene with AAV ITR, the packag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/65C12N15/35C12N7/01
CPCC12N15/86C12N15/65C12N7/00C07K14/005C12N2750/14121C12N2750/14122C12N2750/14143
Inventor 孙秀莲
Owner 济南宜明医疗科技有限公司