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Construction of a shortened autographa californica multiple nucleopolyhedrovirus genome as protein expression vector
Ziqian Cao1, Xinyu Liu1, Jianchao Li1
1College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, P. R. China.
Abstract:
The baculovirus expression system (BEVS) is a powerful tool for protein production in insect cells, and the most widely used virus vector in the BEVS is Autographa californica multiple nucleopolyhedrovirus (AcMNPV), whose genome contains 155 open reading frames (ORFs), nearly half of which are nonessential for viral replication in vitro. Our previous studies have shown that deletion of some large nonessential fragments from the AcMNPV genome can significantly improve the efficiency of production of foreign proteins and that the viral vector can be shortened by more than 10 kb. Using a previously described shortened vector, four additional fragments containing multiple nonessential genes (ac58-61, ac110-114, ac116-119, and ac121-124) were removed from the AcMNPV genome, and the effects on baculovirus replication and foreign protein expression were examined. The results showed that deletion of one of the nonessential fragments did not affect virus replication. For the expression of foreign proteins, removal of ac110-114 and ac116-119 was beneficial for protein production, deletion of ac121-124 apparently did not affect protein production, and deletion of ac58-61 severely impaired protein expression. Simultaneous deletion of two fragments from the AcMNPV genome significantly influenced exogenous protein expression and/or virus replication. Finally, by injection into Spodoptera frugiperda larvae, it was shown that the three shortened AcMNPVs that had maintained their infectivity and ability to express foreign proteins in cultured cells also retained their infectivity in vivo. The three shortened baculovirus expression vectors obtained in this study were 127-129 kb in length, representing a further reduction of 1.4-2.3% in genome size compared to the previously shortened vector, further optimizing it for protein production in the BEVS.
Insights
Shortening the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome by deleting nonessential genes optimizes foreign protein production in the baculovirus expression system (BEVS). Specific gene deletions enhanced protein yield while maintaining viral replication and infectivity in vivo.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- The baculovirus expression system (BEVS) utilizes Autographa californica multiple nucleopolyhedrovirus (AcMNPV) for foreign protein production in insect cells.
- AcMNPV's genome contains numerous nonessential open reading frames (ORFs), offering opportunities for vector optimization.
- Previous work demonstrated that deleting large nonessential fragments improves foreign protein yields and reduces vector size.
Purpose of the Study:
- To investigate the impact of deleting additional nonessential gene fragments (ac58-61, ac110-114, ac116-119, ac121-124) on AcMNPV replication and foreign protein expression.
- To further optimize AcMNPV as a viral vector for enhanced protein production in BEVS.
- To assess the infectivity of modified AcMNPV vectors in vivo.
Main Methods:
- Genetic manipulation of the AcMNPV genome to delete specific nonessential gene clusters.
- Evaluation of baculovirus replication efficiency in insect cells.
- Quantification of foreign protein expression levels.
- Assessment of viral infectivity in Spodoptera frugiperda larvae.
Main Results:
- Deletion of ac110-114 and ac116-119 fragments significantly enhanced foreign protein production.
- Deletion of ac58-61 severely impaired protein expression, while ac121-124 deletion had minimal impact.
- Simultaneous deletions of two fragments affected protein expression and/or viral replication.
- Shortened AcMNPV vectors (127-129 kb) retained infectivity in vitro and in vivo, demonstrating optimized protein production capabilities.
Conclusions:
- Targeted deletion of specific nonessential gene fragments in AcMNPV can significantly enhance foreign protein expression efficiency.
- Optimized AcMNPV vectors maintain essential functions, including replication and infectivity, making them valuable tools for protein production.
- Further genome reduction of AcMNPV vectors provides a more efficient platform for the baculovirus expression system.
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