Re-engineering of bicistronic plasmid pGPD/IFN to construct fusion gene co-expressing Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita (Hamilton) and its in vitro functional analysis
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Title |
Re-engineering of bicistronic plasmid pGPD/IFN to construct fusion gene co-expressing Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita (Hamilton) and its in vitro functional analysis
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Creator |
Valsalam, Anisha
P, Gireesh-Babu Rajendran, Kooloth Valappil Tripathi, Gayatri Bedekar, Megha Kadam |
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Subject |
DNA vaccine
Immune adjuvant pcGPD/IFN pIRES Rohu |
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Description |
204-213
Edwardsiella septicemia disease in the cultured Indian major carps is caused by the fish pathogen Edwardsiella tarda and it is preventable by DNA vaccination. Here, we tried to develop a bicistronic DNA vaccine pGPD/IFN expressing the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita. The vaccine showed high protective efficiency in our previous studies; however as a limitation of bicistronic construct the expression of gene cloned in second frame (B) is poor. To overcome this limitation we re-engineered the construct and designed a fusion gene co-expressing the GAPDH and IFN-γ genes as one frame with an aim to get the optimum expression of both the genes. For this purpose, a fusion insert comprising GAPDH and IFN-γ coding sequences was cloned in to pcDNA3.1(+) plasmid vector. The fusion genes' in vitro expression was confirmed in the striped snakehead fish cell line (SSN-1). Successful expression of the re-engineered fusion gene DNA vaccine in the cell line was achieved at 48h post-transfection, which was confirmed by amplifying the expression transcripts of GAPDH and IFN-γ genes. Thus, the study concludes that the re-engineered fusion vaccine pcGPD/IFN (pcDNA3.1(+) plasmid having fusion GPD/IFN) is functional and can be effectively utilized to vaccinate rohu (Labeo rohita) as it contains the species-specific immune gene (IFN-γ) as an adjuvant. |
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Date |
2023-02-27T08:21:37Z
2023-02-27T08:21:37Z 2023-02 |
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Type |
Article
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Identifier |
0975-1009 (Online); 0019-5189 (Print)
http://nopr.niscpr.res.in/handle/123456789/61420 https://doi.org/10.56042/ijeb.v61i03.62517 |
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Language |
en
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Publisher |
NIScPR-CSIR, India
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Source |
IJEB Vol.61(03) [Mar 2023]
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