Name: production and purification of baculovirus for gene therapy application
Uploaded: 2018-04-09T13:30:00
Description: Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

Este trabalho é apoiado em parte financiando o start-up de Cincinnati infantil Research Foundation (CCRF) M.N. e inovador núcleo Grant (ICG) apoiado pelo centro nacional para Ciências translacionais avançando do National Institutes of Health, sob Prêmio número UL1 TR001425. O conteúdo é exclusivamente da responsabilidade dos autores e não representa necessariamente a opinião oficial do institutos nacionais da saúde.

Veja a Figura 1 para uma ilustração resumindo o protocolo.
1. purificação de Baculovirus plasmídeo
<ol><li>Crescer a cultura bacteriana contendo baculoviral Plasmideo DNA (bacmid)14 em 200 mL de caldo LB com antibióticos; canamicina (50 µ g/mL), tetraciclina (10 µ g/mL) e gentamicina (7 µ g/mL), para 16 h a 37 ° C, em um cenário de agitador orbital a 300 rpm.</li>
	<li>Purificar o bacmid DNA da cultura bacteriana usando lise al...

<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>

O protocolo aqui apresentado descreve a produção de baculovirus no Sf9 células em cultura de suspensão e purificação de baculovirus usando uma cromatografia de afinidade de heparina. Os parâmetros usados neste protocolo maximizar o rendimento e minimizar a inactivação de baculovirus infecciosas. O protocolo fornecido aqui mostra que uma recuperação significativamente melhorada de baculovirus partículas em comparação com as recuperações alcançado por outros9.
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Baculoviridae é usado principalmente para a produção de proteínas recombinantes e vacinas em lepidopteran Spodoptera fugiperda (Sf) 9 células de insetos usando recombinante Autographa californica vírus de poliedrose nuclear multicapsid (AcMNPV) 1 , 2 , 3 , 4. mais recentemente, está emergindo como um vetor promissor para gene terapia aplicação5. Ele é conhecido por ter um amplo acolhimento e tecido tropismo, infecta células quiescentes e proliferação, é não-patogênicas e não integrar o anfitrião cromossoma4,5,6. Além disso, baculovirus pode ser produzida em cultura de suspensão isento de soro que é escalável e permite o sistema fechado de processamento para produção futura Clínica1.
A pureza de baculovirus partículas é importante para a realização eficaz transdução minimizando a citotoxicidade7,8,9. Baculovirus pode ser concentrados por ultracentrifugação ou filtragem de fluxo tangencial (TFF) com impacto limitado na sua infecciosidade. No entanto, estes procedimentos não só concentrar partículas de vírus mas também restos celulares e proteínas de Sf9 cultura, que pode ser tóxico em vitro (observação pessoal) e podem induzir a inflamação ou uma resposta imune quando usado na vivo. Para evitar isso, especialmente quando usando altamente concentradas ações de vírus, baculovirus infecciosas precisa ser purificado e separado da contaminação de partículas.
Vários métodos têm sido relatados para a purificação e concentração de baculovirus vetores10,11,12. As abordagens disponíveis, cromatografia de afinidade de heparina permite um nível elevado de etapa única de purificação com a baixa concentração de proteínas12a contaminar. O método baseia-se na identificação do sulfato de heparan como receptor para baculovirus13,14. Depois de carregar Sf9 sobrenadante de célula na coluna e vinculação de baculovirus, a coluna pode ser lavada com buffer (isotônico) fisiológico para remover as partículas contaminantes frouxamente acopladas ou não acopladas. Desde que a ligação a heparina é reversível, baculovirus partículas podem ser eluídas com um buffer de sal elevado, o que dilui-se imediatamente a concentração de sal (isotônica) fisiológica para evitar inativação por choque osmótica12. Além disso, a produção de baculovirus, bem como captura na e eluição da coluna de cromatografia, pode ser realizada usando um processo do sistema fechado que é compatível com o atual boas práticas de fabricação (cGMP).
Aqui, nós fornecemos um protocolo detalhado para a manufatura, purificação e concentração de baculovirus infecciosas usando centrifugação e cromatografia de afinidade. Brevemente, produzimos baculovirus por transfecção de células Sf9 com um plasmídeo baculovirus em cultura aderente e expandir ainda mais o baculovirus infecciosas na cultura de suspensão livre de soro. Podemos purificar baculovirus usando cromatografia de afinidade de heparina e usar ultracentrifugação como etapa final para altamente concentrado do vetor para a aplicação de terapia de gene.

<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 tem sido tradicionalmente usada para a produção de proteínas recombinantes e vacina. No entanto, mais recentemente, baculovirus está emergindo como um vetor promissor para aplicação de terapia de gene. Aqui, baculovirus é produzido por transfecção transiente o baculovirus do ADN do plasmídeo (bacmid) numa cultura de Sf9 células aderente. Baculovirus posteriormente é expandido em Sf9 células em uma cultura livre de soro suspensão até obter o volume desejado. Em seguida é purificado da cultura sobrenadante usando cromatografia de afinidade de heparina. Sobrenadante de vírus é carregado na coluna de heparina, que se liga a partículas de baculovirus no sobrenadante devido a afinidade de heparina para baculovirus envelop glicoproteína. A coluna é lavada com um buffer para remover contaminantes e baculovirus é eluída da coluna com um buffer de elevado-sal. O eluato é diluído a uma concentração de sal isotônica e baculovirus partículas estão mais concentradas usando ultracentrifugação. Usando esse método, baculovirus pode ser concentrado até 500-fold com uma recuperação de 25% de partículas infecciosas. Embora o protocolo descrito aqui demonstra a produção e purificação do baculovirus de culturas até 1 L, o método pode ser dimensionado-se em uma cultura fechada-sistema de suspensão para produzir um vetor de grau clínico para aplicação de terapia de gene.

O protocolo apresentado é em um diagrama de fluxo (Figura 1). As etapas incluem o transfection transiente de Sf9 células com bacmid DNA para produzir baculovirus em cultura aderente em uma placa, a subsequente amplificação em cultura de suspensão isento de soro, tratamento de nuclease e clarificação por centrifugação e filtração, e a purificação usando a cromatografia de afinidade de heparina seguido de concentração com ultracentrifugação.</p...

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

Produção e purificação de Baculovirus para aplicação de terapia gênica

Neste protocolo, baculovirus é produzido pelo transfection transiente de baculovirus plasmídeo em células Sf9 e amplificado em uma cultura de suspensão livre de soro. O sobrenadante é purificado por cromatografia de afinidade de heparina e mais concentrado por ultracentrifugação. Este protocolo é útil para a produção e purificação de baculovirus para aplicação de terapia de gene.

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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