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Background: On the basis of our previously established single recombinant baculovirus expression vector (BEV)-derived OneBac system, we have optimized the process and expanded the rAAV production range to the full range of serotypes rAAV1-13. Objective: Recombinant adeno-associated virus (rAAV) has been widely used as an efficient transgenic vector in biomedical research, as well as gene therapy. Serotype-associated transduction efficiency, tissue- or cell-type tropism and immunological profile are major considerations in the various applications of rAAVs. There are increasing needs for different serotypes of rAAV, either naturally isolated or artificially engineered. However, affordable and scalable production of a desired serotype of rAAV remains very difficult, especially for researchers lacking relevant experience. Methods: Firstly, the AAV Cap gene was optimized to translate by ribosome leaky scanning and the gene of interest (GOI) was cloned into the pFD/Cap-(ITR-GOI)-Rep2 shuttle plasmid. Following the classical Bac-to-Bac method, sufficient BEV stock containing all rAAV packaging elements can be quickly obtained. Finally, we can repeatedly scale up the production of rAAVs in one week by using a single BEV to infect suspension-cultured Sf9 cells. The rAAV1-13 shows relatively high yields ranging from 5×104 to 4×105 VG/cell. More than 1×1015 VG purified rAAVs can be easily obtained from 5 L suspension-cultured Sf9 cells. Results: As expected, rAAV serotypes 1-13 show different potencies for in vitro transduction and cell-type tropisms. Conclusion: In summary, the single BEV-derived OneBac system should prove popular for laboratory scaling-up production of any serotype of rAAV.

Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for large-scale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 105 vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.

Recombinant adeno-associated virus (rAAV) has been widely used as an efficient transgenic vector in biomedical research, as well as gene therapy. Serotype-associated transduction efficiency, tissue- or cell-type tropism and immunological profile are major considerations in the various applications of rAAVs. There are increasing needs for different serotypes of rAAV, either naturally isolated or artificially engineered. However, affordable and scalable production of a desired serotype of rAAV remains very difficult, especially for researchers lacking relevant experience. On the basis of our previously established single recombinant baculovirus expression vector (BEV)-derived OneBac system, we have optimized the process and expanded the rAAV production range to the full range of serotypes rAAV1-13. Firstly, the AAV Cap gene was optimized to translate by ribosome leaky scanning and the gene of interest (GOI) was cloned into the pFD/Cap-(ITR-GOI)-Rep2 shutte plasmid. Following the classical Bac-to-Bac method, sufficient BEV stock containing all rAAV packaging elements can be quickly obtained. Finally, we can repeatedly scale up production of rAAVs in one week by using a single BEV to infect suspension-cultured Sf9 cells. The rAAV1-13 show relatively high yields ranging from 5×104 to 4×105 VG/cell. More than 1×1015 VG purified rAAVs can be easily obtained from 5 L suspension-cultured Sf9 cells. As expected, rAAV serotypes 1-13 show different potencies for in vitro transduction and cell-type tropisms. In summary, the single BEV-derived OneBac system should prove popular for laboratory scaling-up production of any serotype of rAAV.

Current large-scale recombinant adeno-associated virus (rAAV) production systems based on the baculovirus expression vector (BEV) remain complicated and cost-intensive, and they lack versatility and flexibility. Here we present a novel recombinant baculovirus integrated with all packaging elements for the production of rAAV. To optimize BEV construction, ribosome leaky-scanning mechanism was used to express AAV Rep and Cap proteins downstream of the PH and P10 promoters in the pFast.Bac.Dual vector, respectively, and the rAAV genome was inserted between the two promoters. The yields of rAAV2, rAAV8, and rAAV9 derived from the BEV-infected Sf9 cells exceeded 105 vector genomes (VG) per cell. The BEV was shown to be stable and showed no apparent decrease of rAAV yield after at least four serial passages. The rAAVs derived from the new Bac system displayed high-quality and high-transduction activity. Additionally, rAAV2 could be efficiently generated from BEV-infected beet armyworm larvae at a per-larvae yield of 2.75 ± 1.66 × 1010 VG. The rAAV2 derived from larvae showed a structure similar to the rAAV2 derived from HEK293 cells, and it also displayed high-transduction activity. In summary, the novel BEV is ideally suitable for large-scale rAAV production. Further, this study exploits a potential cost-efficient platform for rAAV production in insect larvae. Keywords: recombinant baculovirus, rAAV production, insect cells and larvae, gene therapy

In this paper, we propose a segmenting method for leaf images taken in the field. Instead of processing images in the whole grayscale range, we choose several subintervals of grayscale to operate on. The subintervals are chosen in accordance with the envelopes of image histograms. The subinterval is composed of random two local minimum points. In order to examine the effect of the method, four evaluation grades are settled as ‘Perfect (P)’, ‘Well (W)’, ‘Average (A)’ and ‘Unsatisfying (U)’. ‘Perfect’ (P) represents the leaf edge segmented is complete; ‘Well’ (W) represents leaf edge segmented is almost complete; ‘Average’ (A) represents there is no complete leaf edge segmented and ‘Unsatisfied’ (U) indicates that the majority of the leaf edge is not segmented from the background. Operating the segmentation on different subintervals and clustering the segmenting results into these four grades, we could obtain the suitable grayscale subintervals; therefore the subintervals chosen before have been optimized. Experiments on four kinds of leaves including jujube, strawberry, begonia and jasmine leaves shows that optimized grayscale interval solutions are sufficient to segment target leaf from the background with high efficiency and accuracy.

According to the meaning of food security, the index system for China's food security was established and the weight on each index was given by adopting AHP method. This paper made measurement and evaluation on the level of China's food security from 2001 to 2007 by using the AHP-GRA method. The results show that the model which conforms to China's actual situation can be used as a food safety pre-warning monitoring. Finally, the paper gives some effective proposals on how to promote China's Grain Security.