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Polyhedrin gene expression on protein production and polyhedra

Shang, Hui
http://orcid.org/0000-0003-2975-8117
http://rave.ohiolink.edu/etdc/view?acc_num=miami1532540645318048

Abstract Details

2018, Doctor of Philosophy, Miami University, Cell, Molecular and Structural Biology (CMSB).
Baculoviruses are invertebrate-specific, double-stranded DNA viruses. They are best known as bio-insecticides for pest control, and as baculovirus expression vector systems (BEVSs) for their versatile applications related to protein production. The molecular foundation for BEVS is that Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of Baculoviridae, displays an exceptionally high-level expression of polyhedrin (polh) gene during insect cell infection. polh is not required for virus infection and replication in vitro. The popular AcMNPV-mediated Bac-to-Bac BEVS consists of a bacmid (a modified AcMNPV genome) and donor vector pFastBacTM. In this system, the open reading frames (ORF) of genes of interest, driven by the polh promoter, are followed by a SV40 polyadenylation (pA) signal in the donor plasmid pFastBacTM. However, we found that when we used this commercial BEVS for viral polh gene expression, its polyhedrin protein yield was only about one third of that of wild-type (wt) AcMNPV. To understand why the Bac-to-Bac BEVS has a lower level of gene expression, and to improve it to get higher protein production levels, we compared the genomes of wt AcMNPV and recombinant strains to examine the effects of the upstream regions of the polh promoter and the pA signal of SV40 on gene expression using reporter genes and gene mapping. We found that an 80-bp cis element 147 bp upstream of the 50-bp polh promoter and a 134-bp polh pA signal are required in pFastBacTM to achieve bacmid protein production levels equivalent to wt AcMNPV in High Five insect cells. The polyhedrin protein plays an important role in using baculoviruses as pesticides, and it is the major structural protein of virion occlusion bodies/polyhedra. Polyhedra protect virions from adverse environments and they are required for oral infection in vivo. A polh ORF DNA fragment was generated by PCR using the wt AcMNPV DNA as the template. A recombinant AcMNPV carrying this polh ORF produced large cuboid polyhedra. By site-directed mutagenesis, scanning and transmission electron microscopy, we identified one novel nucleotide mutation within polh of AcMNPV led to defective virion occlusion and the production of abnormal and larger cuboid polyhedra. Taken together, these findings contribute to a better understanding of polh gene expression and its impact on the morphology of polyhedra and virion occlusion, thereby improving BEVS for higher protein production and engineering baculoviruses for pest control.
Xiao-Wen Cheng (Advisor)
Eileen Bridge (Committee Chair)
Mitchell Balish (Committee Member)
Joseph Carlin (Committee Member)
Susan Hoffman (Other)
117 p.
Microbiology
baculovirus; polyhedrin; protein production; BEVS; polyhedra; virion occlusion; AcMNPV

Recommended Citations

Citations

  • Shang, H. (2018). Polyhedrin gene expression on protein production and polyhedra [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1532540645318048

    APA Style (7th edition)

  • Shang, Hui. Polyhedrin gene expression on protein production and polyhedra . 2018. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1532540645318048.

    MLA Style (8th edition)

  • Shang, Hui. "Polyhedrin gene expression on protein production and polyhedra ." Doctoral dissertation, Miami University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=miami1532540645318048

    Chicago Manual of Style (17th edition)

miami1532540645318048
238
© 2018, some rights reserved.Creative Commons License Polyhedrin gene expression on protein production and polyhedra by Hui Shang is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by Miami University and OhioLINK.
Release 3.2.12