Integration And Expression Of Hu-α-IFN Recombination Gene In Carp Grass

The annual yield of grass carp covers 20% in total output of the fresh water fish,which is fast growing and tastes delicious,besides, the forages of grass carp are various ,with a lower cost of raising,However, at present,the serious diseaes of grass carp results in a lower survival rate 10-15%,which cause more then billions yuan economic losses per year to the fresh water cultiuation ,especially, Hemorrhagic virus of carp lasts for a long period of time and features a high death rate,therefore,it' s a woed-wide problem for the aquacultural scientific researchers to discover an effective settlement ,which is still under the discussion. The rapid development of modem molecular biology technique suppies a possible and efficient way for us to solove the problem completely with a comprehensive researches results to transgenic fish in recent years both domestic and aboard,it proves that it' s possible and makes great senses to cultivate a high quality fish to meet the satisifaction of the people . Making anti-disease breeding researches of fish by transfer gene is a new direction in transfergene researches offish.Hu-α -IFN is a sort of biological activity mass of broad-spectrum antivirus and have evdient effictiveness to varities of viral-disease . it was important glucoprotein modulating cell functions by combination to idiosyncrasy receptor on cell membrane, which inducing process of cascade signal magnifying, then passing signals into nucleus to induce the modulation of the serial gene expression and viarous physiological reaction, and inhence immunity and antivirus. Take advantage of the character of hu- α -IFN, The recombination gene was recombinated hu- α -IFN gene and carp β -actin gene promoter, then it was migrate into the body of lower vertebrate grass carp. Through studing its integration, expression and antivirus, expected to gain a new kind of grass carp assumed notable antivirus of Hemorrhagic virus of carp . the research adopts that Hu- α -IFN gene were introduced into the nuclei of oocytes or cytoplasm of Grass carp to develop anti-disease transgenic grass carp breeding researches,combing the advantage of Hu- α -IFN gene and breeding by Genetic engineering,with an aim of finding out an effective way of solving antivirus of Hemorrhagic virus of carp completely.IN research of transgenic fish , Hu- α -IFN gene ( recombination gene) is cutdown and introduced into the nuclei of oocytes or cytoplasm of Grass carp at one-cell or two-cell stage via micyoinjection by Narashige micyoinjection apparatus . the recombination gene micyoinjected is 5nl DNA (about 106-107) into everyone oocytes or cytoplasm of Grass carp. There were 32197 oocytes of 12 series via micyoinjection and achived 12945 fries in 2000 year , By hatching and raising .Genomic DNA was extracted from Grass carp who were transformated in 1120 Grass carp presented postive reaction by blood . The transgenic Grass carp were detected by PCR , Southern blot. The results showed that PCR applify a 485 specific molecular band in 672 individuals, the rate of positive reaction is 60%, Southern blot result shows a strong signal in transgenic fish . It is concluded that Hu- α -IFN gene has been integrated and expression . It is also that foreign gene integrating position and copy number is different individuals , By ELISA detecting , 23.5% transgenic fish' were detected Hu- α -IFN gene expression in all 672 transgenic fish , but Expression level of Hu-α -DFN is significant difference in different individuals and growth period.it is also conformed that transgenic Grass carp presented masculine in ELISA detecting have more resistivity than control Grass carp in inoculatived GCHV testing , the rate is 60-70% .