Dissertation > Medicine, health > Oncology > Oncology experimental study > Treatment of experimental

Production of Adeno-associated Virus Mediated by Bac-to-Bac Baculovirus Insect Expression System

Author ChenYaNing
Tutor HanShuangYin
School Henan University
Course Oncology
Keywords Baculovirus insect expression system sf9cells adeno-associated virus 293T cells
CLC R73-36
Type Master's thesis
Year 2012
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Objective: To make an efficient and safe method for production of recombinant adeno-associatedvirus (rAAV) of clinical scale by baculovirus insect expression system.Methods: Inverted terminal repeat (ITR) and target gene expression frame of recombinantadeno-associated virus (rAAV) vector pAAV-MCS were digested by restriction endonuclease PciI and SfoI,getting the2053bp of target fragments. The target fragments were purified by gel extraction kit, and thenplained the end of them using Klenow. Baculovirus vector pFastBacDual was digested by restrictionendonuclease KpnI and HindIII, getting the4806bp of target fragments after purification, then the end ofthem were blunted with Klenow and T4Polymerase. Using Solution I ligase, two fragments wereconnected together as the6859bp of cis-acting vector. It was confirmed by double enzyme NdeI and HpaIdigestion and single enzyme EcoRI digestion. After DNA sequence analysis, it was named as pBclAAV-MCS. In order to test the feasibility of rAAV production by baculovirus insect expression system, weamplified the720bp of EGFP gene fragment from the vector pIRES-EGFP using PCR reaction withforward primer EGFP-EcoRI-F and reverse primers EGFP-Xbal-R. As vector pBcl AAV-MCS has the samerestriction enzyme sites EcoRI and XbaI, EGFP gene fragment was inserted into the pBcl of AAV-MCSEcoRI and XbaI sites with Solution I ligase. After DNA sequence analysis, it was named as pBclAAV-EGFP. Based on the leaky scanning mechanism of eukaryotic gene transcription, ACG or CTG wasinserted into several translation initiation, then a promoter can start the expression of multiple fragments atthe same time. After the capsid proteins (Cap1) and replication proteins (Rep2) gene in pAAV-MCS wasamplified by PCR reaction, they were cloned into the sites promoter PP10 and PPH of baculovirus virusexpression vector pFastBacDual. The trans-acting vector was named pBcl AAV Cap/Rep. The cis-actingand trans-acting vector were transformed into DH10Bac competent respectively to make recombinantbaculovirus bacmid, and they were named as Bacmid AAV-EGFP and Bacmid AAV-Cap/Rep afteridentification by PCR correctly. After getting Bacmid DNA with according to Invitrogen plasmid extractionsteps, we transfected them to sf9cells for preparation P1virus with the transfection reagent liposomeCellfectinII respectively, and then used the P1to expand P2virus, and the titers were about1012v.g./ml.rAAV were rescued and packaged by co-infection of P2virus of Bacmid AAV-EGFP and Bacmid AAV-Cap /Rep into insect cells sf9. Finally, we took rAAV infected293T cells to detect its activity.Results: Cis-acting is splicinged together by pAAV-MCS and pFastBacDual, size for6859bp, enzymedigestion and DNA sequencing are all correct, named pBcl AAV-MCS, EGFP is introduced into themultiple cloning site, purpose is to test the feasibility of the system, named pBcl AAV-EGFP sequencing iscorrect; Trans-acting vector is the use of eukaryotic gene expression leakey scanning the packagingcomponents express AAV mechanism, including capsid proteins which are composed of the same mRNA,different initiation codon and common terminator transcription, replication protein contains Rep78andRep52, starting codon respectively is AUG and CUG, will capsid proteins and replication protein the twogene expression of fragments connected to the corresponding enzyme digestion sites by polymerase chainreaction (PCR) reaction, were cloned by baculovirus pFastBacDual vactor of PP10promoter codon of theKpnI and XhoI sites and PPHpromoter codon of HindIII and BamHI sites, by DNA sequencing correctlynamed pBcl AAV Cap/Rep. Than transposition of trans-acting vector and cis-acting into DH10BacTMcompetent cells respectively, turn a form of stem Bacmid, suspension culture in the insects sf9cells intobaculovirus preparation, virus titer to1-3×1012v. g./ml. transposable formed rod Bacmid, in suspensionculture of insect Sf9cells prepared baculovirus, virus titers of up to1-3x1012v.g/ml. Then with the twobaculovirus infected Sf9cells, complete rAAV-EGFP salvation, replication and packaging process, theninfected293T cells test has good activity.Conclusion: The system overcomes the disadvantages of the conventional rAAV preparation ofrestrictive factors, clinical grade virus preparation provides experimental basis for rAAV, gene therapy haslaid a good foundation.

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