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Recombinant adeno-associated virus preparation method and recombinant baculovirus

A technology of recombinant baculovirus and baculovirus, applied in the field of bioengineering, can solve problems such as poor flexibility, low versatility, and complex construction, and achieve the effect of reducing difficulty and enhancing flexibility and versatility

Active Publication Date: 2017-07-04
布林凯斯(深圳)生物技术有限公司
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a preparation method of recombinant adeno-associated virus for gene therapy and a recombinant baculovirus, the purpose of which is to combine the Rep gene, Cap gene and ITR core expression elements of AAV Optimized integration, construction into the baculovirus genome or integration into the host packaging cell genome, to realize the production of rAAV by infecting the host packaging cell line with a recombinant baculovirus, thereby solving the problem of existing large-scale preparation of rAAV methods Complex technical issues with poor flexibility and low versatility

Method used

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  • Recombinant adeno-associated virus preparation method and recombinant baculovirus
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  • Recombinant adeno-associated virus preparation method and recombinant baculovirus

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preparation example Construction

[0031] The preparation method of the recombinant adeno-associated virus provided by the invention comprises the following steps:

[0032](1) The recombinant baculovirus constructed with recombinant adeno-associated virus ITR core expression element and Cap gene or Rep gene is infected with the corresponding host packaging cell line.

[0033] The recombinant baculovirus is used to provide the ITR core expression elements and Cap gene or Rep gene required for the preparation of rAAV, and activate the baculovirus-specific promoter PH or P10 by infecting the host packaging cell line to induce the host packaging cell line to express The Rep gene or Cap gene thus assists the replication and assembly of rAAV.

[0034] The recombinant baculovirus preferably utilizes the pFast.Bac.Dual (pFBD) shuttle vector of the Bac to Bac system (such as figure 1 B) Construction, the specific method is as follows:

[0035] a. The ITR core expression element was cloned into the P10 promoter and PH ...

Embodiment 1

[0057] Embodiment 1: Recombinant baculovirus BEV / Cap-(ITR-GFP) infects Sf9 / Rep packaging cell line to prepare rAAV

[0058] (1) Infect the corresponding host packaging cell line with the recombinant baculovirus constructed with the recombinant adeno-associated virus ITR core expression element and the Cap gene of the corresponding serotype

[0059] The recombinant baculovirus constructed with the recombinant adeno-associated virus ITR core expression element and the Cap gene of the corresponding serotype, that is, the recombinant baculovirus BEV / Cap-(ITR-GFP), was prepared and amplified as follows:

[0060] In order to place the ITR core expression element and the Cap gene required for the preparation of rAAV into a recombinant baculovirus. We utilized the pFast.Bac.Dual (pFBD) shuttle vector in the baculovirus expression system (Bac to Bac) (eg figure 1 B). In the example, the Cap gene based on type 2 AAV was codon-optimized according to the principle of ribosome leakage sc...

Embodiment 2

[0078] Embodiment 2: Recombinant baculovirus BEV / Cap-(ITR-GFP) infects Sf9 / Rep packaging cell line to prepare rAAV

[0079] This embodiment is similar to embodiment 1, the only difference is that the recombinant baculovirus, its main component combination scheme is as follows:

[0080] LinkA-(ITR-GFP)-linkB-CapB

[0081] After infecting the Sf9 / Rep packaging cell line with the recombinant baculovirus BEV / Cap-(ITR-GFP) in this example, the prepared rAAV was purified. The yield of rAAV during the purification process is shown in Table 2. The experimental results show that the rAAV yield of a single Sf9 / Rep packaging cell can reach 7.20×104VG, and after purification by this method, the recovery rate reaches about 31.3%.

[0082] Table 2 Yield analysis of rAAV purification process

[0083]

[0084] HEK293 cells were inoculated into 96-well plates at 1×104cells / well, and infected with purified rAAV at corresponding concentration gradients 6 hours later. After 48 hours of inf...

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Abstract

The present invention discloses a recombinant adeno-associated virus preparation method and recombinant baculovirus. The recombinant adeno-associated virus preparation method specially comprises: (1) infecting a corresponding host packaging cell line with recombinant baculovirus constructed with a recombinant adeno-associated virus ITR core expression element and a Cap gene or Rep gene; (2) carrying out amplification culture on the host packaging cell line infected with the recombinant baculovirus in the step (1) to produce recombinant adeno-associated virus; and (3) separating and purifying the recombinant adeno-associated virus obtained in the step (2), wherein the recombinant adeno-associated virus has the genome constructed with the adeno-associated virus ITR core expression element and the corresponding serotype Cap gene or has the genome constructed with the adeno-associated virus ITR core expression element and the Rep gene. According to the present invention, the flexibility and versatility of the recombinant adeno-associated virus preparation method using the baculovirus system are substantially enhanced.

Description

technical field [0001] The invention belongs to the field of bioengineering, and more specifically relates to a preparation method of a recombinant adeno-associated virus and a recombinant baculovirus. Background technique [0002] Recombinant adeno-associated virus (rAAV) has the characteristics of high safety, low immunogenicity, wide host range, and the ability to mediate long-term stable expression of foreign genes in animals. It is one of the most promising vectors in the field of gene therapy. rAAV plays an important role and has great demand in the fields of neuroscience research and gene therapy of diseases. According to research data, a large primate experiment or clinical gene therapy experiment requires about 1015VG (VG, virus genomes) of rAAV, which is hundreds to thousands of times the amount used in general in vitro cell experiments or mouse experiments (Hum Gene Ther .2002 Nov 1;13(16):1935-43.). [0003] At present, there are two main methods for large-scal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866
CPCC12N7/00C12N15/86C12N2750/14121C12N2710/14043C07K14/005C12N2750/14122C12N2710/14044C12N2750/14143C12N2800/22C12N2710/14022
Inventor 吴阳徐富强何晓斌
Owner 布林凯斯(深圳)生物技术有限公司
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