Encyclopedia of Experiments: Immunology
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Transcript
Chikungunya virus-like particles, VLPs, are non-infectious structures composed of viral envelope proteins embedded within a lipid bilayer, and devoid of genetic material.
To produce Chikungunya VLPs, infect Spodoptera frugiperda Sf9 insect cells with recombinant baculoviruses expressing a Chikungunya structural gene cassette. The cassette consists of a baculovirus promoter fused with chikungunya structural genes encoding capsid, and envelope proteins.
During incubation, baculovirus envelope glycoproteins bind to surface receptors on the insect cells. This facilitates baculovirus entry and release of viral DNA into the cytoplasm, generating polyproteins containing chikungunya capsid and envelope proteins.
Post-synthesis, autocatalytic cleavage of the capsid protein produces the precursor polyprotein possessing envelope proteins in endoplasmic reticulum, ER. In ER lumen, enzymes cleave the precursor polyprotein, generating individual proteins that enter the Golgi compartment for further processing, forming protein heterotrimers.
Golgi resident proteases cleave the protein heterotrimers, producing mature envelope proteins with E2 and E1 proteins. These envelope proteins translocate to the membrane, budding into VLPs and releasing into the medium.
Transfer the infected culture to a tube and centrifuge to pellet the insect cells. Aspirate the VLP-containing supernatant and filter it to remove debris.
The resulting filtrate, containing chikungunya VLPs, is ready for immunopathology studies.
