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Recombinant baculovirus and application thereof

A baculovirus and virus technology, applied in the field of gene therapy, can solve the problems of low virus yield, high production cost, unstable quality, etc., and achieve the effects of high virus quality, improved production capacity, and strong versatility

Active Publication Date: 2017-03-29
布林凯斯(深圳)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above deficiencies or improvement needs of the prior art, the present invention provides a recombinant baculovirus and its application, the purpose of which is to integrate the Rep gene, Cap gene and ITR core expression elements of adeno-associated virus (AAV) As the expression vector of recombinant adeno-associated virus (rAAV) in the virus genome, it solves the problems of poor flexibility, low versatility, complex construction or low virus yield, unstable quality and high production cost in the existing large-scale preparation of rAAV technical issues

Method used

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  • Recombinant baculovirus and application thereof
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  • Recombinant baculovirus and application thereof

Examples

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Effect test

Embodiment 1

[0044] Embodiment 1: Preparation and amplification of recombinant baculovirus (BEV)

[0045] In order to put the three main components required for rAAV preparation: Cap gene, Rep gene and core expression element containing ITR into a recombinant baculovirus. We utilized the pFast.Bac.Dual (pFBD) shuttle vector from the baculovirus expression system Bac to Bac. In the example, the Rep gene based on type 2 AAV was codon-optimized according to the principle of ribosome leak scanning, and the Rep gene was placed under the regulation of the PpH promoter to realize the functional expression of Rep72 and Rep52 replication proteins. The Rep gene sequence is as shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 (the three types correspond to RepA, RepB, ​​and RepC). In the example, the Cap gene based on type 2 AAV was codon-optimized according to the principle of ribosome leak scanning, and the Cap gene was placed under the regulation of the P10 promoter to realize the three capsid pro...

Embodiment 2

[0052] Example 2: Utilize recombinant baculovirus (BEV) to infect Sf9 insect cells to prepare rAAV and verify its activity

[0053] The BEV prepared in Example 1 was used to infect Sf9 cells in suspension culture at a multiplicity of infection (MOI=5). After 72 hours of infection, the cell culture solution was centrifuged at 3000 rpm for 5 min, and the culture supernatant and cell pellet were collected respectively. After BEV is produced, it is mainly released into the supernatant of the medium through secretion, and there are also some unreleased BEVs in Sf9 cells. After rAAV is produced, it mainly exists in the nucleus of Sf9 cells. Since Sf9 cells undergo cytopathic changes (CPE) after infection, some cells will be lysed, and rAAV will also be partially released into the supernatant. Therefore, both BEV and rAAV will be present in the supernatant and cell pellet.

[0054] In order to verify that Sf9 cells were infected with recombinant baculovirus, active rAAV was prepared...

Embodiment 3

[0056] Example 3: Purification and titer determination of rAAV

[0057] Because the rAAV prepared by the three combination schemes in Example 1 is practically the same, the rAAV prepared in Scheme 1 will be used as an example for the subsequent purification and activity detection of the rAAV prepared by the Bac-A system.

[0058] After the recombinant BEV was infected, about 1×10 8 Cells Sf9 cell pellet, add 10ml lysis buffer (50mM Tris-Cl, 150mM NaCl, 2mM MgCl 2 , pH 8.0), followed by repeated freezing and thawing 3 times, centrifuged at 5000rpm for 5min to collect the supernatant, added nuclease Benzonase to the supernatant to a final concentration of 50U / ml, and treated in a water bath at 37°C for 60min. After treatment, centrifuge at 5000rpm for 10min to collect the supernatant. The supernatant was extracted with chloroform, and the supernatant after the extraction was washed with 13.2% (NH 4 ) 2 SO 4 And the solution of 10% PEG8000 carries out the method for two-phas...

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Abstract

The invention discloses recombinant baculovirus and application thereof. The recombinant baculovirus integrates Rep genes and Cap genes of AAVs (Adeno Associated Viruses) and ITR core expression elements. The recombinant baculovirus is applied to preparation of an rAAV (Recombinant Adeno Associated Virus) carrier for gene treatment; the flexibility is high; the universality is high; the virus quality is high; the yield is high; the recombinant baculovirus is applicable to large-scale amplification production; and the difficult problem of rAAV large-scale preparation can be effectively solved.

Description

technical field [0001] The invention belongs to the field of gene therapy, and more specifically relates to a recombinant baculovirus and its application. 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. Research data show that a large primate experiment or a clinical gene therapy trial requires about 10 15 The amount of rAAV of VG (VG, virus genomes) is hundreds to thousands of times that of general in vitro cell tests or mouse tests. Using the traditional method of co-transfecting HEK293 cells with three plasmids, 10 14 The rAAV virus particles of VG need to transfect as many as 5000 more than 175cm 2 C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/864C12R1/93
CPCC12N15/86C12N2710/14143C12N2710/14144C12N2750/14143C12N2750/14152C12N2800/22A61K48/005C12N7/00C12N2710/14044
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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