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Study on Production of Recombinant Adeno-associated Viruses Based on Baculovirus-insect System

Author: LeiQiuGang
Tutor: QianQiJun
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: AAV vectors Baculovirus rAAV2 - EGFP Virus production
CLC: Q789
Type: Master's thesis
Year: 2010
Downloads: 77
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Abstract


Adeno-associated virus (Adeno-associated viruses, AAV) after the infection of the human body does not produce pathogenic recombinant AAV vector retained only the ITR sequences at both ends by removing the viral genes to further increase the security, but also a relatively long period of carrying the gene expression , so more and more people's attention in the field of gene therapy. However, the traditional mode of production is difficult to meet the needs of the clinical AAV vector production, which became a major bottleneck of AAV vectors in clinical applications, and explore an efficient, safe and zoom mode of production in order to get a high yield of AAV The carrier is a necessary condition to meet clinical needs. In addition to the adenovirus and herpes simplex virus, baculovirus for the replication of the AAV packaging auxiliary functions, and the baculovirus is not the same as the adenovirus and herpes simplex virus on the human body toxicity, therefore, the use of baculovirus carrying the AAV cis-and trans-acting elements required for packaging, with baculovirus infected Sf9 insect cells and production of recombinant AAV vectors in insect cells, is an ideal and can meet the clinical needs of novel recombinant AAV production methods. The baculovirus recombinant AAV production system requires two types of trans factors: AAV replication protein Rep78 and Rep52, another type AAV capsid protein Cap1 Cap2 and CAP3 can be carried by two baculovirus or carry by single baculovirus. Another cis components required by the system is a recombinant baculovirus carrying the AAV2 ITR and exogenous expression cassette (EGFP). AAV2 Rep and Cap gene primers were designed, PCR amplification Rep78 is, Rep52, CAP1 and Cap2 3. Rep78 and Cap2 3 group, Rep52, and Cap1 were built into pFastBacDual carrier. In addition, we use a role in insect cells intron Rep and Cap gene construct to the same carrier, with the role of the intron rep expression box at the same time the expression of Rep78 and Rep52 and while Cap expression cassette can expression CAP1 and CAP2. Several of these vectors were transformed into DH10Bac? The competent transposition restructuring restructuring Bacmid, insect cells transfected with the recombinant baculovirus Bac-AAV2Rep78Cap2 3, Bac-AAV2Rep52Cap1 and Bac-AAV2RepiCapi,. A similar approach recombinant baculovirus Bac-AAV2EGFP. BAC-AAV2Rep78Cap2 3, Bac-AAV2Rep52Cap1 and BAC-AAV2EGFP co-infection of Sf9 cells three baculovirus production system, the BAC-AAV2RepiCapi and Bac-AAV2EGFP co-infected Sf9 cells constitute two baculovirus production system, two systems to recombinant AAV2 vector production with EGFP rAAV2-EGFP. Preliminary studies have shown that three baculovirus system and baculovirus expression system can successfully packaged rAAV2-EGFP virus. Virus produced by the AAV Purification Kit yield by quantitative PCR detection of viral vector genome, results showed that: the the three baculovirus production capacity for each Sf9 cells 4.85 × 103VG (Virus vector genomes, viral vector genome), while the two like virus reaches each Sf9 cells, 6.12 × 10 ~ 4VG. Further HEK293 cells after transduction by flow cytometry: the former production capacity for each Sf9 cells 3TU (Transduction Unit transduction units), and the latter about 30TU, and after the infection of HEK293 cells can be seen strong green fluorescent expression, the virus is strong vitality. From the various detection results, the two-baculovirus system more production capacity, output and production efficiency will be greatly improved if the zoom mode of production to large-scale suspension culture. Accordingly, the baculovirus production system efficiency, safety, the zoom back production method greatly simplifies the production process, reduces the production cost, it is possible to satisfy the gene therapy and clinical demand for AAV production.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering) > Application of genetic engineering
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