| Grouper is one of the major marine-cultured fishes in the southern coast of China and in Southeast Asia.Its delicious and tasty meat is high in protein,low in fat,rich in amino acids and vitamins,which are necessary for human beings.All these characteristics make grouper a kind of economical fish with high economic and nutritional values.In recent years,however,with the enlargement of the cultivation scale and the deterioration of local aquacultural environment,the frequency of diseases of Grouper appears to be on rise year by year,which seems to be the biggest obstacle for its mariculture.Therefore,it is significant to conduct a study of the related genes of Groupers' immune mechanism in order to improve the immunity and enhance the disease-resistant ability of Grouper.According to relevant research reports,eIF2 a kinase PKR and HRI in fish is closely related to the immune system of fish.PKR has antiviral ability to activate cells,regulate the immune mechanism of the organism.In this paper,the PKR gene the HRI gene of Epinephelus coioides were cloned and expressed,and their preliminary function were studied.Using RACE technology,the full length cDNA of PKR and HRI genes were cloned from spleen tissues of grouper.The full length of grouper PKR is 2151 bp,including 1863 bp ORF area,encoding 621 amino acids.Its protein molecular weight is 70157.15 Daltons,PI is 5.72.The full length of HRI gene is 2467 bp,including 1995 bp ORP area,encoding 664 amino acids.Its protein molecular weight is 74207.58 Daltons,and PI is 6.23.BLAST comparison on the NCBI web site found that its PKR and HRI highly homologous to those of other species.By constructing prokaryotic expression vector PKR-pET-32a and HRI-pET-32a,the fusion protein of PKR and HRI was obtained and purified.The mouse antiserum of PKR was also prepared.The result of tissue distribution for the HRI and PKR showed that,the HRI and PKR were expressed in all tissues of group.The PKR gene expression is highest in intestines and stomach,and HRI expression is highest in spleen,intestine and stomach.The subcellular localization of PKR was also studied and the results showed that PKR distributed in all the cells but mainly in the cytoplasm.Under heat shock pressure,HRI was express in unprocessed GS cells to some extant,and the expression level of HRI increased gradually after heat shock treatment and reached its peak after 2 to 3 hours.Then HRI expression level fell down.After stimulated by inactivated virus,the HRI and PKR expression level in grouper GS cells raise,in which the PKR expression level reach the peak after stimulated by VNN for 12h and the PKR expression level reach the peak after stimulated by SGIV for 48h.Results of dual luciferase reporter gene experimental testing showed that the PKR and HRI could activate nf-kappa B report gene to different degree,and activation effect of PKR is stronger than HRI.The results of this study showed that eIF2 alpha kinase PKR and HRI played important roles in host immune response against viral infections.Not only did this study enrich the achievement of immune response mechanism,but also provides a new strategy and theoretical basis basis for virus disease contol in the further. |
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