## 2 Insect Cell-Baculovirus Expression Vector System
### Development of Baculovirus Vectors  
Numerous baculovirus vector systems employ homologous recombination in insect cells to introduce foreign genes into viral genomes. The initial baculovirus vector, BacPAK6, originates from a modified circular (infectious) AcMNPV genome. The vector undergoes a \"triple-cut\" process via Busa36I digestion, leading to its linearization and the removal of one _lacZ_ gene and two essential gene fragments (ORF603 and ORF1629). This modification renders the viral DNA incapable of replicating in insect cells. Subsequently, linearized viral DNA and a transfer plasmid containing foreign genes are transfected into insect cells, facilitating homologous recombination and the insertion of foreign genes into the viral genome. This process restores the ORF and infectivity through allelic replacement of circular viral DNA, \begin{table}
\begin{tabular}{c c c c} \hline \hline
**System** & **Advantage** & **Disadvantage** & **References** \\ \hline \multirow{4}{*}{Yeast system} & High production efficiency, low cost, & Protein modification between yeast and mammals is quite different, which affects the infectivity of the virus. The efficiency of virus assembly and packaging is low, and it is still in the research stage. & [33; 34] \\  & Protein modification is consistent with the human body and can produce high-efficiency rAAV particles. Mature technology, wide application, suitable for laboratory research and small-scale production. & [35; 36] \\  & High yield, low cost, suitable for large-scale production, mature production process, suitable for industrial production. & The early process development is complex, the early investment is high, and the production environment is demanding. & [37; 38] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Different systems for production of rAAV. culminating in the generation of recombinant budded viruses via genome replication. Despite the simplicity and minimal requirements of homologous recombination, Bsu36l digestion's efficiency is not consistently 100%, resulting in a mixture of recombinant and the original parent virus after the first replication cycle. Consequently, the isolation of the recombinant virus from the parent virus becomes necessary, a task achieved through plaque purification (distinguishing white recombinant plaques from blue parent plaques). This step typically demands substantial labor, technical expertise, and time [43; 44] (Figure 2A). To streamline the intricate process of plaque purification of the recombinant virus, a shuttle bacmid containing a Tn7 attachment site and propagating in _Escherichia coli_ (_E. coli_) is introduced from a transfer vector to facilitate the translocation of foreign genes, which is called pFastBac vector. This modification significantly simplifies the manufacturing process of recombinant baculovirus, as the bacmid can be efficiently amplified and modified in _E. coli_ and remains infectious in insect cells. Among them, the polyhedron promoter (pPh) allows high-efficiency and high-level expression of recombinant protein in insect cells. Tn7L and Tn7R allow site-specific transpositions of the gene of interest to Mini Tn7 elements in baculovirus genome. Site-specific transposition takes place between the bacmid genome and the transfer vector upon introducing the transfer vector into _E. coli_ containing the AcMNPV bacmid. This results in the insertion of the target gene into the pPh site, disrupting the _lacZ_ sequence. After identifying recombinant bacilli, they undergo expansion based on antibiotic resistance and blue/white selection on agar plates before being isolated and introduced into insect cells to produce bacterial bacilli. Although this system eliminates the need for plaque purification, several necessary and somewhat intricate steps must be completed before recombinant viruses in _E. coli_ can proceed into insect cells [45; 46] (Figure 2B). Additionally, a dual-functional transfer vector, pFastBacDual, is available.