1. Chikungunya virus-like particles (VLP) Expression Using Baculovirus/Insect Cell System
<ol>
	<li>Generate recombinant baculovirus expressing chikungunya virus capsid and envelope proteins (C-E3-E2-6K-E1) from S-27 strain using a commercial baculovirus expression system.</li>
	<li>Culture&#160;Spodoptera frugiperda&#160;Sf9 cells in serum-free Sf9 growth medium with 100 U/ml penicillin and 100 &#181;g/ml streptomycin at 28 &#176;C. Use 3-5 x 105&#160;c/ml to initiate spinner flask cultures for suspension cell growth. Passage routinely when they reach cell densities of 2-4 x 106&#160;c/ml (every 3-4 days).</li>
	<li>Culture Sf9 cells in suspension in spinner flasks with stirring at 130 rpm on a multipoint stirrer plate system. For proper aeration, maintain culture volumes at no more than half the volume of the spinner flask.</li>
	<li>For expression of Chikungunya or CHIK VLPs, infect Sf9 cells at a density of 2 x 106&#160;cells/ml with recombinant baculovirus at a multiplicity of infection of 1 and return to 28 &#176;C incubator. Typically, infect one or two spinner flasks, containing 250 ml Sf9 cells. 
	Note: While we do not use this method, Sf9 cells may be cultured in shaker flasks at 28 &#176;C using a shaker platform.</li>
	<li>Harvest cultures after 72-96 hours post-infection, or when cell viability has decreased to 70-80% as determined by Trypan Blue Exclusion according to the manufacturer's protocol. 
	Note: The cells continue to proliferate while infected and there is a ~80% viability in Sf9 cultures infected with recombinant CHIK VLP baculovirus at 3 days post-infection (dpi). Baculovirus-infected cells are larger in appearance. Morphology may also change from round to oblong. In the late stages of baculovirus infections, the cells began to lyse.</li>
	<li>Transfer cultures directly from suspension culture into 50 ml conical tubes and spin the cells down at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Collect supernatants and filter through a 0.22 &#181;m pore membrane before sedimentation via ultracentrifugation.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Opti-MEM I</td>
			<td>Life Technologies</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus Expression System</td>
			<td>Life Technologies</td>
			<td>10359-016</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m vacuum filter top (500 mL)</td>
			<td>Nalgene</td>
			<td>569-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf-900 II SFM</td>
			<td>ThermoFisher Scientific</td>
			<td>10902-096</td>
			<td></td>
		</tr>
		<tr>
			<td>Cimarec I 6 multipoint stirrer plate</td>
			<td>ThermoFisher Scientific</td>
			<td>50094596</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of chikungunya virus-like particles using baculovirus expression system in insect cells

Generation of Chikungunya Virus-Like Particles Using Baculovirus Expression System in Insect Cells

Source: Arevalo, M. T. et al., <a href='https://app.jove.com/v/54041'>Expression and Purification of Virus-like Particles for Vaccination.</a>&#160;J. Vis. Exp. (2016)
This video shows how virus-like particles, VLPs are generated by infecting insect cells with a recombinant baculovirus carrying the chikungunya virus gene cassette. The recombinant virus infects cells, leading to VLP formation, assembly, and release into the culture medium for potential use in vaccine production.

Source: Arevalo, M. T. et al., Expression and Purification of Virus-like Particles for Vaccination.&#160;J. Vis. Exp. (2016)
This video shows how ...

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.

Culture previously prepared Spodoptera frugiperda, or Sf9 cells, in suspension in spinner flasks by continuously stirring at 130 rpm on a multipoint stir plate system. Maintain culture volumes at no more than half the volume of the spinner flask for proper aeration. Next, express CHIK VLPs by infecting 250 milliliters of Sf9 cells in a spinner flask at a density of 2 x 106 cells per milliliter with previously prepared recombinant baculovirus, and return the cells to a 28-degree Celsius incubator.
Use trypan blue exclusion to determine if the cell viability has decreased to 70% to 80%. Upon confirmation, transfer the cultures directly from suspension into 50-milliliter conical tubes, and spin the cells down. Collect the supernatants, and filter them through a 0.22-micron pore membrane before sedimentation.

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1.0K Views. 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,...

Generation of Chikungunya Virus-Like Particles Using Baculovirus Expression System in Insect Cells

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Tags

Name	Company	Catalog Number
Opti-MEM I	Life Technologies	31985062
Bac-to-Bac Baculovirus Expression System	Life Technologies	10359-016
0.22 μm vacuum filter top (500 mL)	Nalgene	569-0020
Sf-900 II SFM	ThermoFisher Scientific	10902-096
Cimarec I 6 multipoint stirrer plate	ThermoFisher Scientific	50094596