What is baculovirus expression?
A baculovirus expression vector (BEV) is a recombinant baculovirus with a double-stranded circular DNA genome that has been genetically modified to include a foreign gene of interest. BEVs are viable and can infect susceptible hosts, usually cultured lepidopteran insect cells or larvae, in a helper-independent fashion.
What is baculovirus system?
The baculovirus expression vector system (BEVS) has become an established commercial manufacturing platform. The authors review this technology, which enables fast, flexible and scalable protein production and is increasingly being used for the production of viral vaccines and gene therapy vectors.
Why is baculovirus used?
Baculovirus is extensively utilized as an excellent tool for production of recombinant protein in insect cells. Baculovirus infects insects in nature and is non-pathogenic to humans. In addition to insect cells, baculovirus is capable of transducing a broad range of animal cells.
How will you develop baculovirus vector?
Baculovirus vectors are generally produced by homologous recombination between a shuttle vector and a wild-type genome, using the viral polyhedrin gene as the recombination target.
How does baculovirus expression system work?
The BaculoDirect baculovirus expression system (Invitrogen) uses the Gateway technology to permit the direct transfer of a gene of interest into the baculovirus genome, without the need for production of recombinant bacmid DNA.
Can baculovirus infect human cells?
Despite the restricted host, it was reported that baculovirus can infect human cells (Volkman and Goldsmith, 1983).
What is the baculovirus genome?
Baculovirus genomes are covalently closed circles of double stranded-DNA varying in size between 80 and 180 kilobase-pair. The genomes of more than fourty-one baculoviruses have been sequenced to date.
Which are the baculovirus types?
Nuclear Polyhedro… VirusCydia pomonella granulovirus
Baculoviridae/Lower classifications
How do you calculate Moi from PFU ml?
PFU is the Virus titer (Virus per ml). MOI is the ratio between the number of viral particle and the number of cells. So basically, MOI= PFU/cell.
What are the advantages of insect expression systems?
Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus.
Is baculovirus a biocontrol agent?
Most of the Baculoviruses are the genus of Nucleopolyhedrovirus, which are used as biological control agents. They are pathogenic, attacking insects and such arthropods.
Does baculovirus integrate?
Baculovirus transduction of mammalian cells is transient in nature as the foreign DNA does not integrate into the host genome.
Is baculovirus enveloped?
Baculoviruses are a family of enveloped, large double-stranded DNA viruses that predominantly infect insects.
What does an MOI of 0.1 mean?
Answer: Multiplicity of infection (MOI) refers to the number of viral particle(s) present relative to host cell(s). For example, if one million viral particles are added to one million cells, the MOI is 1.0. If 100,000 viral particles are added to one million host cells, the MOI is 0.1.
How is MOI calculated?
MOI stands for Multiplicity of Infection which refers to the number of viral particles per cell. To calculate, take the number of viral particles used per well then divide by the number of cells originally seeded in the well. This equals the MOI.
Why we use BL21 for protein expression?
The BL21(DE3)pLysS competent cells provide tighter control of protein expression for expression of toxic proteins and are resistant to chloramphenicol. When used with the CE6 bacteriophage, the BL21 cells provide the tightest control of protein expression (see BL21(DE3) Strains and Protein Toxicity).
What are the advantages of protein expression in insect cells?
Insect cells offer high levels of protein expression with posttranslational modification approaching that of mammalian cells, ease of scale-up, and simplified cell growth that can be readily adapted to high-density suspension culture for large-scale expression.
How do insect cells purify proteins?
Protein insolubility, following recombinant expression in insect cells, can occur. However, using the methods described herein, it is possible to extract and purify insoluble protein using affinity, ion-exchange and gel filtration chromatography. Indeed, protein insolubility often aids protein purification.
What is the optimal MOI?
According to Table 1, the optimal MOI to use is 2. So, if you purchase 50 microliters of viral particles and the titer is 108 TU/ml, then you should have a total of 0.05 ml x 108 particles ml-1 = 5 x 106 particles.
What happens if MOI is too high?
If MOI is too high, the efficiency of gene transfer may not be very high, but many copies of transgene may integrate into the chromosomes of the target cells instead, thus causing chromosomal instability [24].
What does MOI 10 mean?
Multiplicity of infection (MOI) is a frequently used term in virology which refers to the number of virions that are added per cell during infection. If one million virions are added to one million cells, the MOI is one. If ten million virions are added, the MOI is ten.
Why are BL21 E. coli used?
E. coli BL21 has been routinely used for non-T7 expression, and it was also recently modified to produce a plasmid DNA vaccine, due to its better performance in high-cell-density fed-batch cultures compared to K-12 strains (2).
What is the difference between BL21 and BL21 DE3?
BL21(DE3)pLysS is a derivative of BL21 that has the T7 RNA polymerase gene under the control of the lacUV5 promoter. This arrangement is on a phage genome, called DE3. DE3 is inserted into the chromosome of BL21 to make BL21(DE3). pLysS is a plasmid that contains the T7 lysozyme gene (LysS).
How do you purify baculovirus?
Baculovirus supernatant is treated with a nuclease, centrifuged, and filtered. Baculovirus is purified by heparin affinity column chromatography, diluted in buffer, and concentrated aseptically by ultracentrifugation.