A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

Abstract

BackgroundGarlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors.ResultsIn this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR.ConclusionsThe expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

“…Since then, thousands of recombinant proteins have been expressed in insect cells for numerous different applications using different strategies [21,22]. Furthermore, baculovirus could be also used as biological control agents.…”

A Novel Betabaculovirus Isolated from the Monocot Pest Mocis latipes (Lepidoptera: Noctuidae) and the Evolution of Multiple-Copy Genes

“…This is the first description of a polyhedral and not cuboidal crystals formed by a Granulin replacing the polyhedrin gene in AcMNPV. A single non-synonimous mutation in polyhedrin has been shown to be able to change drastically the crystal morphology and the ability to occlude virions [ 45 , 47 , 48 ]. We do not know why the expression of MolaGV granulin in Sf21 cells produced polyhedral-shaped OBs instead of the cuboidal-shaped OBs produced by recombinant BmNPV in Bm2 cells [ 39 ] and recombinant AcMNPV in Trichoplusia ni (BTI-TN-5B1-4) cells [ 40 ].…”

Expression of 1B capsid protein of Foot-and-mouth disease virus (FMDV) using baculovirus expression system and its validation in detecting SAT 2- specific antisera

“…Reasons for using BacSF9 system for protein expression included (1) secretion into the growth medium; (2) production of less aggregated proteins with an increased probability of correct folding, and (3) post-translational modifications for better antigenicity and specificity. Previously, the baculovirus expression system was successfully used to express different viral proteins such as the tomato yellow leaf curl virus coat protein (TYLCV) (El-Gaied, Salem & Elmenofy, 2017), Garlic mite-borne filamentous virus (GarMbFV) (Ardisson-Arau'jo et al, 2013), and glycoprotein E (gE) of the Egyptian BoHV-1.1 Abu-Hammad strain (rBac/gE-AbuH) (El-Kholy et al, 2013;El-Gaied et al, 2020). Furthermore, recombinant proteins produced using this system were validated for their antigenicity.…”