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. 2011 Jul;3(3):64-70.

Effective Genetic Expression of Nanoantibodies by Recombinant Adenoviral Vector in vitro

I Yu Gribova  1 , S V Tillib, I L Tutykhina, Capital Em Cyrillic М Shmarov, D Yu Logunov, L V Verkhovskaya, B S Naroditskii, A L Gintsburg
Affiliations

Effective Genetic Expression of Nanoantibodies by Recombinant Adenoviral Vector in vitro

I Yu Gribova et al. Acta Naturae. 2011 Jul.

Abstract

The present study is devoted to the feasibility of expressing the single-domain mini-antibody (nanoantibody) selected from the library of sequences of the variable domains of special single-stranded antibodies derived from an immunized camel, a gene of which was introduced into eukaryotic cells within a recombinant adenoviral vector. A vector bearing the gene of a single-domain nanoantibody was obtained using the AdEasy Adenoviral Vector System (Stratagene). This method of delivering the nanoantibody gene facilitates efficient expression of this gene and functional activity of the nanoantibody. The results obtained can be used to produce passive immunizing tools against pathogens or new-generation immunobiological antitoxic medication.

Keywords: genetic immunization; nanoantibodies; recombinant adenoviral vector.

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Figures

Fig. 1
Fig. 1
Schematic description of the genetic constructions used. A – Genetic construction containing the gene of nanoantibody against cytokeratin. B – The recombinant adenovirus genome bearing the gene of nanoantibody against cytokeratin.
Fig. 2
Fig. 2
The analysis of anti-cytokeratin nanoantibody expression in cells infected with recombinant adenovirus. A – Expression of anti-cytokeratin nanoantibody gene in cells infected with recombinant adenovirus Ad5-aCyK-VHH was analyzed using reverse transcription (RT)-PCR, cDNA encoding this gene was amplified by PCR with primers specific to the gene of the anti-cytokeratin nanoantibody (aCyK-VHH), Ad5 fiber gene (Fib Ad5), and house-keeping gene GAPDH. A recombinant adenovirus with no transgenic insertions in the E1 deletion region of the adenoviral genome (Ad-null) was used as the specificity control. B – The expression of the anti-cytokeratin nanoantibody was detected by hybridization with anti-HA antibodies in a Western blot analysis. A protein with a molecular weight of 15 kDa was detected in the cultural fluid of cells infected with the recombinant adenovirus. The (His 6 )-tagged nanoantibody produced in E. coli was used as the control of the specificity of the interaction between anti-HA antibodies and the target protein.
Fig. 3
Fig. 3
Western blot detection of the functional activity of nanoantibodies. Total cell extracts of liver (lanes 1, 3) and brain (lanes 2, 4) cells were separated on a SDS–PAGE and electrophoretically transferred to a PVDF-membrane. The specific interaction of the target protein (~ 55 kDa) with the anti-cytokeratin nanoantibodies obtained in the cultural fluid of cells infected with the recombinant adenovirus and periplasm of E. coli was detected by immunoblotting.
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References

    1. Deyev S.M., Lebedenko E.N.. Acta Naturae. 2009;1(1):32–50. - PMC - PubMed
    1. Stern M., Herrmann R.. Critical Rev. Oncol./Hematol. 2005;54:11–29. - PubMed
    1. Robinson C.R., Sauer R.T.. Proc. Natl. Acad. Sci. USA. 1998;95:5929–5934. - PMC - PubMed
    1. Worn A., Pluckthun A.. J. Mol. Biol. 2001;305:989–1010. - PubMed
    1. Hamers-Casterman C., Atarhouch T., Muyldermans S., Robinson G., Hamers C., Songa E.B., Bendahman N., Hamers R.. Nature. 1993;363:446–448. - PubMed

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