Construction of a DNA Vaccine Using Glycoprotein Gene and Its Expression Towards Increasing Survival Rate of KHV-Infected Common Carp ( Cyprinus carpio )

Deoxyribonucleic acid (DNA) vaccine has recently been developed as an alternative vaccine against virus infection. This study was the first step of DNA vaccine development to protect cyprinids including common carp ( Cyprinus carpio ) and fancy koi ( Cyprinus carpio ) from KHV (koi herpesvirus) infection in Indonesia. One of KHV glycoprotein genes, i.e. glycoprotein (GP) was ligated with Japanese medaka ( Oryzias latipes ) â-actin promoter to generate pAct/GP as a DNA vaccine. Fourty fish in body weight of 10-15 g/fish were individually injected by pAct/GP into muscle in different dosage of 2.5 µg, 7.5 µg and 12.5 µg/100 µl phosphate buffer saline. Total RNA was extracted from the 12.5 µg of pAct/GP-injected fish muscle at 24, 48 and 67 hours post-injection to analyze GP expression by RT-PCR method. Potential of pAct/GP as DNA vaccine was examined by injecting KHV into the 30-days-vaccinated fish. Both of possitive and negative control fish group were not vaccinated. Possitive control fish group were injected with KHV, but negative control fish group were not. KHV-challenged fish were reared for 1 month, and the death fish were calculated daily. Result of RT-PCR analysis showed that GP gene expression were detected at 3 d post-injection. Expression of GP in the vaccinated fish groups helped to improve their survival rate after challenged by KHV. All of fish without DNA vaccination had dead 17 days after KHV injection. The results demonstrated that pAct/GP had high potency to be used as a DNA vaccine against KHV infection in cyprinids.


INTRODUCTION
DNA vaccination is the direct inoculation of DNA expression vector, such as plasmid containing a specific gene from viruses driven by eukaryotic promoter, to stimulate the in vivo synthesis of immunogenic proteins and immune responses (Hirono, 2005). DNA vaccine has some advantages, such as activating both of humoral and cellular mechanisms, good effect when given at an early stage, and inducing protection shortly after vaccination at both low and high water temperature (Lorenzen & LaPatra 2005).
As a poikiloterm organism, immune responses of fish are influenced by environmental water temperature (Vinitnantharat et al., 1999). Water temperatureindependent activity of DNA vaccine may meet the need to induce immune response in order to overcome mass mortality of fish as virus infection. Further, protection of attenuated vaccine is also highly on both humoral and cellular responses. However, attenuated vaccine may cause auto-infection. In contrary, DNA vaccine is most likely far away to be auto-infection agent. In DNA vaccination, just a part of virus genes is introduced to fish (Hirono, 2005).
As an alternative to chemical and antibiotic treatment, a DNA vaccine is the efficacious method for the prevention of infectious diseases in farmed fish, especially viral infectious diseases, because it can not result in anti-medication of fish and the pollution of water (Heppel & Davis, 2000;Zheng et al., 2006). DNA vaccine can induce strong and longlasting humoral and celldependent immune responses and posses several practical and economical advantages, such as without  (Tanghe et al., 2000). Immunization with plasmid DNA vector represents a promising new approach to vaccination. It has been shown to elicit humoral and cellular immunity and protection in various infection models (Mollenkopf et al., 2004). This vaccine may be applicated in carp farming to KHV infection.
Protective immunity against IHNV (Infectious hematopoietic necrosis virus) had been achieved through of both non-specific and specific immune responses by vaccination with plasmid DNA encoding a viral glycoprotein (G) (Kim et al., 2000). The distribution and expression of lymphocystis disease virus (LCDV) vaccine, on the basis of DNA vaccine (pEGFP-N2-LCDV0.6 kb) construction, were analyzed by RT-PCR. Results from RT-PCR studies indicated that the Mcp gene is expressed in all tissues of vaccinated fish 7-20 hours after vaccination, therefore may have provided an antigen producing specific immune response (Zheng et al., 2006). This observations prompted an investigation into the potency of the glycoprotein to be used as a DNA vaccine and it's dose improving carp resistance to KHV infection. Results of partial sequencing read from forward direction were showed with following figure.

MATERIALS AND METHODS
Results of GP25 similarity analysis with data in GenBank were 99% similar with KHV from Japan, USA and Israel. Results of GP allignment with data sequence at GenBank were showed with following figure 3.
Results of sequences analysis showed that GP gene that had been used as an insert DNA to pGEMT-Easy was glycoprotein target of koi herrpesvirus (KHV).
Protein production using glycoprotein DNA had protected fry up to 80 d after immunization and induced protective neutralizing antibodiess (Corbeil et al., 1999).

CONCLUSIONS
Plasmid of pAct-GP has high potency to be used as a DNA vaccine improving carp resistance to KHV infection. Vaccination of fish using 12.5 ìg pAct-GP gave highest survival rate after challenge test with lethal dose of KHV compared to 2.5 and 7.5 µg.