Immunoprotection Of Grass Carp (Ctenopharyngodon Idella) With Recombinant Interferon RCiIFN

Grass carp (Ctenopharyngodon idella) is among the top-ranking of freshwater fish species in terms of aquaculture production in the world, while the aquaculture production of grass carp in China accounts for95.7percent of the world’s total. However, intensive grass carp farming in China has suffered badly from grass carp haemorrhage, an epidemic and fatal disease caused by the Grass Carp Haemorrhage Virus (GCHV), resulting in a high mortality rate and an enormous economic loss to the aquaculture industry. The use of antibiotics and chemotherapeutics in large quantities to control fish diseases inevitably leads to resistance of the pathogens and pollutes the environment. Commercial vaccines are showing some promise in tackling the problems, but do have the disadvantage in their strict specificity against particular pathogens. Furthermore, teleosts are a group of poikilothermal vertebrates and their innate immune systems play major roles in the defense against viral infections. Administration of recombinant fish IFN is one of the most efficacious methods of maintaining a healthy fish population and is a promising alternative to the use of antibiotics and vaccines.On the basis of our previous successful cloning of a type I IFN in grass carp, a recombinant plasmid pET32a(+)-CiIFN was constructed in the present study, and then transformed into E. coli BL21(DE3) plysS. We established a stable system of prokaryotic expression and purification of grass carp recombinant IFN (rCiIFN). Highly purified and bioactive rCiIFN protein was harvested by affinity chromatography using Ni-NTA His-Bind Resin. As determined by SDS-PAGE, the rCiIFN fusion protein is about38kD, the rCiIFN protein concentration was750μg/mL using Bradford assay. Cytopathic effect inhibition assay was used to examine the titer of rCiIFN, which was equivalent to5.7×103U/μg. Rabbit polyclonal antibodies was prepared using purified rCiIFN, its titer (>50000) was measured by indirect ELISA assay. MTT reduction assay was used to examine the immunoprotective effects of rCiIFN against GCHV infections. The results indicated that the bioactivities of rCiIFN were also dosage-dependent.The immunostimulatory effect and in vitro or in vivo antiviral activities of rCiIFN were investigated in the present study. To evaluate in vivo protective efficacy of rCiIFN against grass carp hemorrhagic virus (GCHV) infection, we administered it by intraperitoneal (ip) injection and oral feeding in our experiments. Intraperitoneal injections with three different rCiIFN dosages (0.1,1,10μg/g fish body weight) at24h prior to GCHV infection all improved the survival rate of the fish. The relative percentage survival (RPS) from1μg/g fish body weight injection was higher than those from other two dosages. Fish were fed with pellets mixed with rCiIFN at1μg/g fish bod}’weight once daily for1,2and5days before GCHV infection. The results indicated that the RPS from feeding for2consecutive days was higher than those from feeding for just1d or5consecutive days.Additionally, we observed that several IFN-stimulated genes (ISGs) were greatly up-regulated. After stimulation with rCiIFN. the expression levels of Mx and IFN genes in grass carp kidney cells (CIK) were highly enhanced and peaked within6and12h, respectively. By comparison with gene expression levels observed in PBS-injected controls, expressions of IFN, IRFL IRF7. PKR, Mx and STAT3were found to be significantly up-regulated in tested tissues of rCiIFN-injected fish, and most of these ISGs reached the highest level within12h post-treatment and declined within72h. After feeding fish once daily for1-5d with pellets mixed with rCiIFN at1μg rCiIFN/g fish body weight, it was found that the expression level of IFN was significantly up-regulated and reached its peak level in tissues of those fish treated for1or2d.The purified rCiIFN protein was intubated orally into the alimentary tract of grass carp at the dosage of2μg/g fish body weight. Six hours later, frozen sections of the anterior and posterior segments of grass carp intestine were prepared, and immunohistochemical technique was applied to examine the absorption abilities of intact bioactive rCiIFN in different segments of intestine. The results showed that the absorption potential of intact rCiIFN was higher in the posterior intestine than in the anterior intestine.In addition, the pharmacokinetics of rCiIFN in grass carp plasma was determined by a sandwich ELISA. Grass carps were ip injected or intubated with purified rCiIFN at the dosage of0.75μg/g fish body weight, the peak levels of rCiIFN in grass carp plasma was observed at2h and3h, with the respective concentration of159.17pg/mL and79.06pg/mL, and then rapidly decrease to the constitutive levels within24h. These characteristics of pharmacokinetics of rCiIFN in grass carp plasma were in accordance with the first-order absorption as well as the first-order clearance process.A desk study of the practicability of rCiIFN application was also carried out in this study. Highly purified rCiIFN protein was obtained as a result of the denaturation and renaturation treatment of the inclusion bodies which were formed in the recombinant bacteria. As shown in a pilot test, rCiIFN could be steadily reduced when the recombinant bacteria was cultured in a5L fermenter. Analysis of adsorption and dissolution assay indicated that pellet could adsorb rCiIFN tightly, and the loss of the pellet-adsorbed rCiIFN was very small after the pellets were dissolved in PBS within a short period of time.