| Turbot, Scophthalmus maximus, as a traditionally farmed food fish species in the world. Since it was introduced to China by Yellow Sea Fisheries Research Institute in 1992, and cultured vastly in the Jiaodong Peninsula, Shandong Province, from 1998 to now , turbot has become the most important aquaculture species in coastal areas of northern China. However, a novel virus, turbot reddish body iridovirus ( TRBIV ) caused a new kind of epizootic disease"Viral reddish body syndrome"(VRBS) in farmed turbot in 2001, which is belonging to the genus meglocytivirus of the family Iridoviridae, and it has caused severe mortalities of farmed turbot and led to serious economic losses in China since then. Consequently, VRBS was one of the most difficult facters for the sustaining development of turbot culture because there was no any method to control the Propagation of TRBIV in vivo up to now.The auther investigated the epidemics of TRBIV from 2005 to 2007, basing on the conclusions drew by Prof. Shi Cheng-yin at 2004. Epidemic investigation of TRBIV showed that thousands of infected fish generally lacked external lesions. The only consistent abnormalities were pale gills with local haemorrhages , petechial haemorrhages in fins and fin bases , especially haemorrhages in muscleand skin. Anaemia , pallor and enlarged kidneys were usually observed in severe affected fish. However, there were farmed turbot dead with some new sympotoms, such as: pale body color, an enlarged abdomen, protruding eyes and so on. Investigation also showed that farmed turbot had lower rate of mortality when they lived under water tempreture 18°C. In the epidemic investigation, we also find that higher desity about farmed tubort will aggravate the disease in the farmed turbot.Using a pair of primers that designed from the highly conserved region of the major capsid protein gene of TRBIV, a PCR product of 781bp was attained with the DNA template of the farmed turbots that had obvious clinical signs of VRBS caused by TRBIV. TRBIV was detected in these tissuses, such as: brain, gill , heart, liver, digestive system , spleen , kidney , blood and the gonad of the parent turbot. However, no virus was detected in the muscle interesting. These results implied that the virus lied vertical transmission through the epidemic investigation. So it is necessary to study vertical transmission of TRBIV by other methods or technologies in the future. But TRBIV was also detected in the turbots, which had pale body color, an enlarged abdomen and protruding eyes, or no any clinical signs about VRBS, and these living under water tempreture 18°C. These results implies that TRBIV can hide in the body of tubort and and destroy the immune system of turbot, lastly result in the death of infected turbot with the accumulation of virus particles.In this study, we designed PCR primers which eliminate the signal pepetide in order to be expressed properly. With the vector pUCm-T/TRBIV MCP as template, we can get the PCR products TRBIV MCP 1373 bp. After digestion with endonuclease, the PCR products were ligated into eukaryotic expression vector pGAPZαA, and then transformed into E.coli Top10F'by electroporation, and the recombinant vectors pGAPZαA/TRBIV MCP were achieved.After the recombinant vectors were corrected by PCR, digestion with endonuclease, and sequence, the recombinant vector pGAPZαA/TRBIV MCP was digested by AvrⅡand transformed into Pichia pastoris X-33 by electroporation. At first, ZeocinTM was used to screen the recombinant yeast clones. Secondly the recombinant yeasts were identified by PCR with the recombinant yeast genosome as template. Then the higher expressional recombinant yeasts were detected by Elisa-blot. We picked out seven of the identified yeasts to culture at 30°C, and ferment at 28°C, and collect the supernatant by centrifuge lastly, Then the total proteins were obtained. The above proteins were detected by SDS-PAGE and Western-blot, and a protein at the size of 54KD was signal showed. That result implied that the recombinant TRBIV MCP was expressedsuccessfully. |
