抗草鱼出血病病毒转基因稀有鮈鲫的初步研究

RESISTANCE OF SIGCRV TRANSGENIC RARE MINNOW(GOBIOCYPRIS RARUS) TO GRASS CARP REOVIRUS

研究采用草鱼H1基因启动子,以草鱼呼肠孤病毒(Grass carp reovirus,GCRV)外衣壳蛋白VP7基因为靶基因,以增强型绿色荧光蛋白(eGFP)为报告基因,构建了3个小发卡RNA(shRNA)表达载体pH1siGCRV(x)-CMVeGFP。CIK细胞感染实验表明,pH1siGCRV2-CMVeGFP具有较高的病毒抑制作用。通过显微注射将pH1siGCRV2-CMVeGFP导入稀有鮈鲫(Gobiocypris rarus)受精卵,获得转基因稀有鮈鲫P0代群体。转基因稀有鮈鲫攻毒实验显示,转基因稀有鮈鲫死亡率为30%,抗草鱼出血病能力显著提高。进一步的实时荧光定量PCR检测证实,转基因稀有鮈鲫脾脏、后肠和肝脏中GCRV的含量显著低于对照鱼,并随着时间的延续逐渐减少,转基因稀有鮈鲫体内GCRV的复制受到有效抑制。研究为抗草鱼出血病转基因鱼育种奠定重要基础。

In this study,three shRNA expression vectors pH1siGCRV（x）-CMVeGFP were constructed by employing grass carp H1 promoter,using outer capsid protein VP7 gene of grass carp reovirus（GCRV） as target gene and eGFP as report gene.CIK cells transfected experiment suggested that pH1siGCRV2-CMVeGFP had the strongest suppression on GCRV replication.Transgenic P0 group of fish were obtained by microinjecting pH1siGCRV2-CMVeGFP to rare minnow（Gobiocypris rarus）embryos.The transgenic fish had only 30% mortality after GCRV infection.It was shown that the resistance of the transgenic fish to GCRV has significantly improved.Moreover,the results of real-time PCR on VP7 gene in spleen,hindgut and liver confirmed that the content of GCRV in transgenic fish was significantly lower than control and gradually reduced over time,indicating that the replication of GCRV has been suppressed in transgenic fish.This research has provided a solid foundation for producing GCRV-resistant transgenic fish