ABSTRACT
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-?) 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 adjuvant
ABSTRACT
The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K+-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35‰ salinities and maintained for 20 days to observe long term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5‰ compared to 25‰ (isosmotic salinity) and 0.48 folds lower at 35‰. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5‰ the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25‰ and 35‰ NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25‰. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5‰ measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.