Production and Purification of Baculovirus for Gene Therapy Application

Summary

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.

Abstract

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.

Introduction

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 chromosome

Protocol

See Figure 1 for an illustration summarizing the protocol.

1. Purification of Baculovirus Plasmid DNA

1. Grow bacterial culture containing baculoviral plasmid DNA (bacmid)¹⁴ in 200 mL of LB broth with antibiotics; kanamycin (50 µg/mL), tetracycline (10 µg/mL), and gentamycin (7 µg/mL), for 16 h at 37 °C on an orbital shaker setting at 300 rpm.
2. Purify the bacmid DNA from the bacterial culture using standard alkaline lysis protocol¹⁵, and dissolve the bacmid in TE buffer.

2. Production of Baculo....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

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