Valsalam, AnishaP, Gireesh-BabuRajendran, Kooloth ValappilTripathi, GayatriBedekar, Megha Kadam2023-08-092023-08-092023-03Valsalam Anisha, P Gireesh-Babu, Rajendran Kooloth Valappil, Tripathi Gayatri, Bedekar Megha Kadam. 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-?) gene of Labeo rohita (Hamilton) and its in vitro functional analysis. Indian Journal of Experimental Biology. 2023 Mar; 61(3): 204-2130975-10090019-5189http://imsear.searo.who.int/handle/123456789/222588Edwardsiella 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-?) 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-? 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-? 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-? 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-?) as an adjuvantDNA vaccineImmune adjuvantpcGPD/IFNpIRESRohuJournal ArticleIndiaDepartment of Aquatic Animal Health ManagementDepartment of Fish Genetics and Biotechnology, ICAR- Central Institute of Fisheries Education, Mumbai 400 061, IndiaPresent Address: ICAR- National Research Centre on Meat, Hyderabad-500 092, India