This work is supported in part by the Start-Up funding from Cincinnati Children's Research Foundation (CCRF) to M.N. and Innovative Core Grant (ICG) supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1 TR001425. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

See Figure 1 for an illustration summarizing the protocol.
1. Purification of Baculovirus Plasmid DNA
<ol>
	<li>Grow bacterial culture containing baculoviral plasmid DNA (bacmid)14 in 200 mL of LB broth with antibiotics; kanamycin (50 &#181;g/mL), tetracycline (10 &#181;g/mL), and gentamycin (7 &#181;g/mL), for 16 h at 37 &#176;C on an orbital shaker setting at 300 rpm.</li>
	<li>Purify the bacmid DNA from the bacterial culture using stan...

<ol>
	<li>Ikonomou, L., Schneider, Y. J., Agathos, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+cell+culture+for+industrial+production+of+recombinant+proteins.">Insect cell culture for industrial production of recombinant proteins.</a> Appl Microbiol Biotechnol. 62 (1), 1-20 (2003).</li><li>Contreras-Gomez, A., Sanchez-Miron, A., Garcia-Camacho, F., Molina-Grima, E., Chisti, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+using+the+baculovirus-insect+cell+expression+system.">Protein production using the baculovirus-insect cell expression system.</a> Biotechnol Prog. 30 (1), 1-18 (2014).</li><li>Cox, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+vaccines+produced+in+insect+cells.">Recombinant protein vaccines produced in insect cells.</a> Vaccine. 30 (10), 1759-1766 (2012).</li><li>Boyce, F. M., Bucher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus-mediated+gene+transfer+into+mammalian+cells.">Baculovirus-mediated gene transfer into mammalian cells.</a> Proc Natl Acad Sci U S A. 93 (6), 2348-2352 (1996).</li><li>Airenne, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus:+an+insect-derived+vector+for+diverse+gene+transfer+applications.">Baculovirus: an insect-derived vector for diverse gene transfer applications.</a> Mol Ther. 21 (4), 739-749 (2013).</li><li>Sung, L. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+delivery+into+cell+lines+and+stem+cells+using+baculovirus.">Efficient gene delivery into cell lines and stem cells using baculovirus.</a> Nat Protoc. 9 (8), 1882-1899 (2014).</li><li>Zeng, J., Du, J., Lin, J., Bak, X. Y., Wu, C., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transient+transduction+of+human+embryonic+stem+cell-derived+neurons+with+baculoviral+vectors.">High-efficiency transient transduction of human embryonic stem cell-derived neurons with baculoviral vectors.</a> Mol Ther. 17 (9), 1585-1593 (2009).</li><li>Zeng, J., Du, J., Zhao, Y., Palanisamy, N., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculoviral+vector-mediated+transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25 (4), 1055-1061 (2007).</li><li>Wu, C., Soh, K. Y., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+membrane+chromatography+method+for+rapid+and+efficient+purification+of+recombinant+baculovirus+and+baculovirus+gp64+protein.">Ion-exchange membrane chromatography method for rapid and efficient purification of recombinant baculovirus and baculovirus gp64 protein.</a> Hum Gene Ther. 18 (7), 665-672 (2007).</li><li>Grein, T. A., Michalsky, R., Vega Lopez, M., Czermak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+a+recombinant+baculovirus+of+Autographa+californica+M+nucleopolyhedrovirus+by+ion+exchange+membrane+chromatography.">Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography.</a> J Virol Methods. 183 (2), 117-124 (2012).</li><li>Nasimuzzaman, M., Lynn, D., van der Loo, J. C., Malik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+baculovirus+vectors+using+heparin+affinity+chromatography.">Purification of baculovirus vectors using heparin affinity chromatography.</a> Mol Ther Methods Clin Dev. 3, 16071 (2016).</li><li>Makkonen, K. E., Turkki, P., Laakkonen, J. P., Yla-Herttuala, S., Marjomaki, V., Airenne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-o-+and+N-sulfated+syndecan-1+promotes+baculovirus+binding+and+entry+into+Mammalian+cells.">6-o- and N-sulfated syndecan-1 promotes baculovirus binding and entry into Mammalian cells.</a> J Virol. 87 (20), 11148-11159 (2013).</li><li>Nasimuzzaman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+in+baculovirus+binding+and+entry+of+mammalian+cells.">Heparan sulfate in baculovirus binding and entry of mammalian cells.</a> J Virol. 88 (8), 4607-4608 (2014).</li><li>Urabe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+generation+of+high-titer+recombinant+adeno-associated+virus+type+5+in+insect+cells.">Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells.</a> J Virol. 80 (4), 1874-1885 (2006).</li><li>Feliciello, I., Chinali, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+alkaline+lysis+method+for+the+preparation+of+highly+purified+plasmid+DNA+from+Escherichia+coli.">A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli.</a> Anal Biochem. 212 (2), 394-401 (1993).</li><li>Nasimuzzaman, M., Persons, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Membrane-associated+heparan+sulfate+is+a+receptor+for+prototype+foamy+virus+in+human,+monkey,+and+rodent+cells.">Cell Membrane-associated heparan sulfate is a receptor for prototype foamy virus in human, monkey, and rodent cells.</a> Mol Ther. 20 (6), 1158-1166 (2012).</li><li>Earl, P. L., Americo, J. L., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+a+vaccinia+virus+neutralization+assay+based+on+flow+cytometric+detection+of+green+fluorescent+protein.">Development and use of a vaccinia virus neutralization assay based on flow cytometric detection of green fluorescent protein.</a> J Virol. 77 (19), 10684-10688 (2003).</li><li>Kim, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+tumor+growth+in+xenograft+model+mice+by+programmed+cell+death+4+gene+delivery+using+folate-PEG-baculovirus.">Suppression of tumor growth in xenograft model mice by programmed cell death 4 gene delivery using folate-PEG-baculovirus.</a> Cancer Gene Ther. 17 (11), 751-760 (2010).</li><li>Stanbridge, L. J., Dussupt, V., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculoviruses+as+vectors+for+gene+therapy+against+human+prostate+cancer.">Baculoviruses as vectors for gene therapy against human prostate cancer.</a> J Biomed Biotechnol. 2003 (2), 79-91 (2003).</li><li>Nasimuzzaman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+high-titer+foamy+virus+vector+for+the+treatment+of+leukocyte+adhesion+deficiency.">Production and purification of high-titer foamy virus vector for the treatment of leukocyte adhesion deficiency.</a> Mol Ther Methods Clin Dev. 3, 16004 (2015).</li></ol>

The authors declare no conflict of interest.

The protocol presented here describes the production of baculovirus in Sf9 cells in suspension culture and purification of baculovirus using a heparin affinity chromatography. The parameters used in this protocol maximize the yield and minimize the inactivation of infectious baculovirus. The protocol provided here shows a significantly improved recovery of baculovirus particles as compared to recoveries achieved by others9.
Due to broad host range and tissue tropism, se...

Baculovirus is primarily used for the production of recombinant proteins and vaccines in lepidopteran Spodoptera fugiperda (Sf)9 insect cells by using recombinant Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV)1,2,3,4. More recently, it is emerging as a promising vector for gene therapy application5. It is known to have a broad host and tissue tropism, infects both quiescent and proliferating cells, is non-pathogenic, and does not integrate into the host chromosome4,5,6. Moreover, baculovirus can be produced in serum-free suspension culture which is scalable and allows for closed system processing for future clinical production1.
The purity of baculovirus particles is important for achieving effective transduction while minimizing cytotoxicity7,8,9. Baculovirus can be concentrated by ultracentrifugation or tangential flow filtration (TFF) with limited impact on its infectivity. However, these procedures not only concentrate virus particles but also cellular debris and proteins from Sf9 culture, which can be toxic in vitro (personal observation) and may induce inflammation or an immune response when used in vivo. To avoid this, especially when using highly concentrated virus stocks, infectious baculovirus needs to be purified and separated from contaminating particles.
Several methods have been reported for the purification and concentration of baculovirus vectors10,11,12. Of the available approaches, heparin affinity chromatography allows for a single-step high level of purification with the low concentration of contaminating proteins12. The method is based on the identification of heparan sulfate as the receptor for baculovirus13,14. After loading Sf9 cell supernatant onto the column and binding of baculovirus, the column can be washed with physiologic (isotonic) buffer to remove unbound or loosely bound contaminating particles. Since the binding to heparin is reversible, baculovirus particles can be eluted with a high salt buffer, which is diluted immediately to physiologic (isotonic) salt concentration to prevent inactivation by osmotic shock12. Moreover, the production of baculovirus, as well as capture on and elution from the chromatography column, can be performed using a closed-system process which is compatible with current good manufacturing practices (cGMP).
Here, we provide a detailed protocol for the manufacture, purification, and concentration of infectious baculovirus using affinity chromatography and centrifugation. Briefly, we produce baculovirus by transfection of Sf9 cells with a baculovirus plasmid DNA in adherent culture and further expand the infectious baculovirus in serum-free suspension culture. We purify baculovirus using heparin affinity chromatography and use ultracentrifugation as the final step to highly concentrate the vector for gene therapy application.

<table border="0" cellpadding="0" cellspacing="0" style="width:784px;" width="784">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="16">
			<td height="16" style="height:16px;width:257px;">Akta Avant 150</td>
			<td style="width:204px;">GE Healthcare</td>
			<td style="width:156px;">28976337</td>
			<td style="width:167px;">Chromatography system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:257px;">POROS Heparin 50 &#181;m Column</td>
			<td>ThermoFisher Scientific</td>
			<td>4333414</td>
			<td>Heparin column</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td>Non-catalog item</td>
			<td>Concentrates virus at high-speed</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Polyallomer Ultracentrifuge tube</td>
			<td>Beckman-Coulter</td>
			<td>326823</td>
			<td>Concentrates virus at high-speed</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MaxQ 8000 orbital shaker incubator&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>Non-catalog item</td>
			<td>Shaker for suspension culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mL Erlenmeyer flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238071</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L Erlenmeyer flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238072</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microscope (Olympus CKX41)</td>
			<td>Olympus</td>
			<td>Non-catalog item</td>
			<td>Cell monitoring and counting&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Table top centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75253839/433607</td>
			<td>For clarification of Baculovirus supernatant</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">50 ml Conical tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td>For collection of Baculovirus supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>07-200-80</td>
			<td>Tissue culture treated plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10-cm plate</td>
			<td>ThermoFisher Scientific</td>
			<td>08-772E</td>
			<td>Tissue culture treated plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Stericup-HV, 0.45&#160;&#181;m, PVDF</td>
			<td>EMD-Millipore</td>
			<td>SCHVU05RE</td>
			<td>Filtration unit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15160-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracycline</td>
			<td>Sigma-Aldrich</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gentamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bac-to-Bac Vector Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>10360-014</td>
			<td>Baculovirus expression system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DH10B-T1R&#160; Competent cell</td>
			<td>ThermoFisher Scientific</td>
			<td>12331-013</td>
			<td>Competent cell for bacmid</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">TE buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>10 mM Tris-HCl, 1 mM EDTA, pH 8.0.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid Maxiprep kit</td>
			<td>ThermoFisher Scientific</td>
			<td>K2100-06</td>
			<td>For bacmid purification</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sf9 Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>11496-015</td>
			<td>Insect cells</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Grace&#8217;s Insect Cell Culture Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11605-094</td>
			<td>Transfection medium</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>20012227</td>
			<td>Washing cells, diluting samples</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:257px;">HyClone SFX-Insect cell media</td>
			<td>GE Healthcare</td>
			<td>SH30278.02</td>
			<td>Serum-free insect cell growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>Enzyme to degrade DNA and RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Baculovirus plasmid (bacmid) DNA</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>Bacmid for Baculovirus Production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellfectin II&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>10362</td>
			<td>Transfection reagent for insect cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4737</td>
			<td>Stabilizes Baculovirus</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryovial</td>
			<td>Thomas Scientific</td>
			<td>1222C24</td>
			<td>For storage of Baculovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HT1080 cell line</td>
			<td>ATCC</td>
			<td style="width:156px;">CCL-121</td>
			<td>Fibroblast cell line</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Growth media for cell lines</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wash buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elution buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Column cleaning buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile water</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>1.09137</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7073</td>
			<td>For Akta Avant cleaning</td>
		</tr>
	</tbody>
</table>

production and purification of baculovirus for gene therapy application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

The protocol presented is in a flow diagram (Figure 1). Steps include the transient transfection of Sf9 cells with bacmid DNA to produce baculovirus in adherent culture in a plate, the subsequent amplification in serum-free suspension culture, nuclease treatment and clarification by centrifugation and filtration, and the purification using the heparin affinity chromatography followed by concentration with ultracentrifugation.
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Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

Production and Purification of Baculovirus for Gene Therapy Application

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

The overall goal of this protocol is to produce baculovirus in insect cell culture, purify with heparin affinity chromatography and to concentrate with ultracentrifugation. The final product can be used for in vivo gene transfer or as a vaccine. This method can help researchers who want to use purified and concentrated baculovirus for gene therapy application.Baculovirus particles may contain cellular debris and proteins from Sf9 culture which may induce inflammation or an immune response when used in people. To avoid toxicity, baculovirus is purified and separated from contaminating particles. Some parts of the procedure will be demonstrated by Marilou Narciso, a research assistant from my laboratory.To set up the suspension culture, seed 50 milliliters of Sf9 cells in insect culture medium into a 250 milliliters polycarbonate Erlenmeyer flask. And place the flask in an orbital shaker incubator. Add two milliliters of P2 baculovirus stock to the cells, and shake at 135rpm while maintaining a constant temperature of 28 degrees Celsius.Four days after infection, the entire Sf9 cell population will show cytopathic effect, which is an indication of high titer baculovirus production. Sequentially amplify the baculovirus until the desired volume is obtained, by increasing the volume of cell culture in each subsequent infection 10 to 50-fold. After centrifuging the final baculovirus supernatant for 30 minutes at 1000g and four degrees Celsius to separate the course cellular debris, transfer the supernatant to a clean vessel and treat with nuclease at 50 units per milliliter for two hours at 37 degrees Celsius to digest the genomic DNA and RNA.Finally, filter the viral supernatant through a 0.45 micron filtration unit. Set up the chromatography system by placing the wash buffer line from inlet port A1 into a bottle containing at least 500 milliliters of wash buffer. Use the A2 buffer inlet port for loading the elution buffer.Insert several 50 milliliters conical tubes into the fraction collector to collect the column pass-through baculovirus supernatant, the wash buffer, and the eluded baculovirus. Equilibrate the heparin column with five 7.9 milliliters column volumes of wash buffer at a linear flow rate of seven milliliters per minute. Load 250 milliliters of baculovirus supernatant, onto the heparin column using the sample pump of the inlet sample port at a linear flow rate of two milliliters per minute.Then run 10 column volumes of wash buffer through the heparin column at a linear flow rate of two milliliters per minute until the ultraviolet absorbance curve has returned to baseline and become stable. Elude the baculovirus particles from the heparin column with five column volumes of elution buffer at a linear flow rate of four milliliters per minute. Watch for a sharp elution peak of protein on the chromatogram when the baculovirus particles dissociate from the heparin column.Post elution, immediately dilute the eluded baculovirus supernatant 10-fold using 20 millimeter sodium phosphate buffer to prevent an activation of baculovirus particles from osmotic shock during subsequent centrifugation. Begin the concentration procedure by adding an equal volume of the baculovirus supernatant to autoclave to ultracentrifuge tubes. Measure the tube weight by a balance and adjust the weight of the contents of the ultracentrifuge tubes with sterile PBS.Place each of the ultracentrifuge tubes in an opposing bucket of the SW 32 Ti rotor and run the ultracentrifuge at 80, 000g for 90 minutes at four degrees Celsius. Following centrifugation, open the ultracentrifuge tubes in a tissue culture hood and aseptically aspirate the liquid without removing the baculovirus pellet. Add 200 microliters of PBS containing 0.5%BSA and resuspend each pellet by vigorously pipetting up and down.Finally, transfer 50 to 100 microliter aliquots of the concentrated baculovirus to cryovials and store at negative 80 degrees Celsius. Baculovirus titers were estimated by infection of HT1080 cells. The line connected by diamonds shows the total infectious units of baculovirus in each fraction.Baculovirus particles strongly bind to the heparin column, therefore no significant amount of virus is detected in the column wash buffer. A sharp peak of protein is observed during elution, which corresponds to the baculovirus fraction. The parameters used in this protocol maximize the yield and minimize the inactivation of infectious baculovirus particles.This protocol can be used for purification of other viruses and proteins that have an affinity for the heparin.

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14.4K Görüntüleme.  Cincinnati Children's Hospital Medical Center. The overall goal of this protocol is to produce baculovirus in insect cell culture, purify with heparin affinity chromatography and to concentrate with ultracentrifugation. The final product can be used for in vivo gene transfer or as a vaccine. This method can help researchers who want to use purified and concentrated baculovirus for gene therapy application.Baculovirus particles may contain cellular debris and proteins from Sf9 culture which may induce inflammation or an immune respo...