Capability Test of Viral Nervous Necrosis DNA Vaccine Candidate in Antibody Induction of Humpback Grouper (Cromileptes altivelis).

Abstract

Viral nervous necrosis (VNN) is one of the diseases that often infect grouper fish. Two DNA vaccines for VNN, namely pmBA-CP and pJfKer-CP have been constructed. The pmBA- CP construct is controlled by medaka β-actin (mBA) promoter, while pJfKer-CP by Japanese flounder keratin (JfKer) promoter. This study was aimed to examine the antibody induction of coat protein (CP) DNA vaccinated humpback grouper (Cromileptes altivelis). The research was divided into three steps, i.e. (1) analysis of the CP VNN using SDS-PAGE and Western blot methods, (2) analysis of CP mRNA expression using reverse transcriptase PCR (RT-PCR) method, and (3) analysis of anti VNN antibody induction of vaccinated fish using enzyme linked immunosorbent assay (ELISA) sandwich method, and VNN challenge test of vaccinated fish. The results of SDSPAGE showed a specific protein of about 42 kDa from DNA vaccine-injected catfish embryos. By Western blot analysis using a polyclonal antibody produced in this study, the similar size of protein was also obtained in catfish embryos injected by DNA vaccine. This suggests that the polyclonal antibody anti VNN from rabbit immunization with VNN could recognize CP protein from DNA vaccine injected catfish embryos. In subsequent experiment, we used pmBA-CP DNA construct due to the result of CP protein from pmBA-CP injected catfish embryos was higher than that of pJfKer-CP. Humpback grouper juvenile was intramuscularly injected with pmBA-CP in a dose of 12.5 μg per fish, and RTPCR analysis were performed to detect the present of CP mRNA in the fish muscle extracted at different time post vaccination. The results showed that CP mRNA expression was detected at 24 hours postvaccination. This means that mBA promoter could regulate the expression of CP gene in humpback grouper. Furthermore, the result of ELISA showed higher ratio of S/P using serum from pmBA-CP vaccinated fish than unvaccinated fish (control group) at 21 to 35 days postvaccination. This suggests that DNA vaccination is capable of inducing the antibody anti VNN in humpback grouper. Furthermore, the result of challenge test with VNN virus titer of 103,5 FID50/0,2 ml generated 60% relative percent survival. Thus, pmBA-CP DNA vaccination can be useful to improve the resistance of grouper fish against VNN infection, and support the increasing aquaculture production level.