Organizing co mmittee

 
 

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Poster presentation 
185 
OBTAINING RECOMBINANT BACULOVIRUSES
IN Bac-to-Bac® PLATFORM 
 
Sh.Sh. Khasanov, M.Sh. Shukurjonov, J.M. Abdurakhmanov, O.N. Ashirov,
F.B. Eshboev, S.A. Gaynazarova, S.A. Sasmakov, Sh.S. Azimova 
 
S.Yu. Yunusov Institute of the Chemistry of Plant Substances Academy of sciences of the 
Republic of Uzbekistan st. Mirzo-Ulugbek, 77, 100170 Tashkent 
e-mail: [e-mail] 
Usually, for construction of recombinant baculoviruses for highly efficient 
expression of a foreign gene the cDNA of this gene is ligated with plasmid DNA 
containing a viral DNA fragment. Also, the target cDNA is inserted under the control of 
a strong viral polyhedrin gene promoter [1]. To obtain a recombinant baculovirus the 
insect cells are infected simultaneously with the recombinant plasmid and wild-type 
baculovirus. Recombination between homologous sequences of viral and plasmid DNA 
leads to the replacement of the polyhedrin gene in wild-type viral DNA with plasmid 
sequences containing foreign cDNA [2]. Today, these processes cause several 
difficulties. We have chosen the relatively easy-to-use Bac-to-Bac® platform. The Bac-
to-Bac® Baculovirus Expression System provides a rapid and efficient method to 
generate recombinant baculoviruses. This method is based on site-specific transposition 
of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in 
Escherichia coli. 
In our experiments, we obtained a recombinant baculovirus (bacmid) using the 
Escherichia coli
host strain DH10Bac™ and the pFastBac™ genetic construct 
containing a baculovirus vector (bacmid) and a helper plasmid. We generated 
recombinant baculoviruses using the MAX Efficiency® DH10Bac™ chemically 
competent cells (Transform Aid Bacterial Transformation Kit). The LB agar plates 
containing 60 μg/ml kanamycin, 7 μg/ml gentamicin and 15 μg/ml tetracycline used to 
select DH10Bac™ recombinant transformants. To verify the presence of gene of 
interest in the recombinant bacmid the PCR assay is performed. The size of obtained 
recombinant bacmid DNA is greater than 135 kb. 
References: 
1. BacPAK

Baculovirus Expression System User Manual. Cat. No. 631402 
PT1260-1(072313), 2013. http://www.clontech.com 
2. O′Reilly D.R., Miller L.K., Luckow V.A. Baculovirus Expression Vectors: A 
Laboratory Manual. Oxford University Press, 1994, 347 pp.