In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate.
| Autor: | Ishak J; Research Center for Biotechnology, Faridabad, India., Unsunnidhal L; Research Center for Biotechnology, Faridabad, India., Martien R; Department of Pharmaceutics, Faridabad, India., Kusumawati A; Research Center for Biotechnology, Faridabad, India.; Department of Reproduction and Obstetrics, Faculty of Veterinary Medicine, University Gadjah Mada, Yogyakarta, 55281, Indonesia. |
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| Jazyk: | angličtina |
| Zdroj: | Journal of veterinary research [J Vet Res] 2019 Mar 22; Vol. 63 (1), pp. 7-16. Date of Electronic Publication: 2019 Mar 22 (Print Publication: 2019). |
| DOI: | 10.2478/jvetres-2019-0018 |
| Abstrakt: | Introduction: The development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex. Material and Methods: Cloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection. Results: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent. Conclusion: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro . This initial study will be used for further improvement and evaluation in vivo . Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article. |
| Databáze: | MEDLINE |
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