UPTAKE AND BIO-DISTRIBUTION OF GFP EXPRESSION PLASMID IN Penaeus vannamei POST-LARVAE DELIVERED THROUGH IMMERSION ROUTE
White Spot Syndrome Virus (WSSV) is one of the devastating diseases in the shrimp industry. DNA vaccine immunization in the form of genetically engineered plasmid DNA (pDNA) to target viral genes could be a promising approach to control this menace. Immersion is the most practical route of administration for DNA vaccines in shrimp culture. However, it is essential to determine the uptake and bio-distribution of the plasmid DNA delivered through immersion route. The dynamics of plasmid uptake and bio-distribution in vivo can best be showed using a fluorescent reporter that can be visualized under a fluorescence microscope. Green fluorescent protein (GFP) is popularly used as reporter for various biological applications.
In this study, a plasmid DNA construct (pCMV-EGFP) constitutively expressing GFP was developed to track the uptake and bio-distribution in shrimp post-larvae (PL) post-immersion delivery (Fig. 1). First, the SSN1 cells (>80% confluency) were transfected with pCMV-EGFP construct (500 ng) and GFP expression was confirmed after 24h by fluorescence microscopy. After the in vitro validation, the pCMV-EGFP construct was delivered to Penaeus vannamei PL-20 (~15mg) @ 10µg/g body weight through immersion for 30 min.
Visual observation of plasmid uptake and its bio-distribution was studied at 1, 2, 3, 4 and 5 days post-treatment by fluorescence microscopy. GFP expression could be seen in shrimp PL dip-treated with pCMV-EGFP construct while there was no expression in control group (Fig. 2). GFP expression was observed in muscle and telson regions of PL suggesting that these are the two major routes of pDNA entry during immersion treatment. The expression of GFP was tracked till 5 days post-treatment and it was observed that the green fluorescence decreased over time. The present study highlights the possible use of immersion as an effective vaccine delivery route in shrimp post-larvae.
