对虾传染性皮下及造血组织坏死病毒编码蛋白的相互作用研究  

INTERACTIONS BETWEEN INFECTIOUS HYPODERMAL AND HEMATOPOIETIC NECROSIS VIRUS(IHHNV) ENCODING PROTEINS OF PENAEID SHRIMP

作　　者：陈沈雪 魏永伟[1] 苗亮[1] 陈炯[1] 

机构地区：[1]宁波大学海洋学院生物化学与分子生物学实验室,宁波315211

出　　处：《海洋与湖沼》2016年第1期205-212,共8页Oceanologia Et Limnologia Sinica

基　　金：国家高技术研究发展计划(863计划)项目;2012AA020101号;浙江省重大科技专项计划项目;2013C03045-1号;宁波市自然科学基金项目;2014A610182号

摘　　要：采用Matchmaker酵母双杂交系统,将对虾传染性皮下及造血组织坏死病毒(Infection hypodermal and hematopoietic necrosis virus,IHHNV)编码蛋白NS1、NS2和CP序列分别构建到酵母猎物载体p GADT7和诱饵载体p GBKT7上,分别转化至酵母AH109以检测重组猎物载体和诱饵载体的自激活作用及对宿主的毒性作用,发现无自激活作用和毒性作用,随后将重组猎物载体和诱饵载体两两共转至酵母AH109中,涂布于SD/-Leu/-Trp固体培养基上,再点种至SD/-Ade/-His/-Leu/-Trp/X-α-gal固体培养基以鉴定编码蛋白间的相互作用。表型鉴定结果显示,只有共转化有p GADT7-CP/p GBKT7-CP的酵母重组子能够在SD/-Ade/-His/-Leu/-Trp/X-α-gal上生长并显蓝,而其它重组子均不能在其上生长,表明病毒的CP能够自身互作,而其他编码蛋白间无相互作用。为了进一步研究病毒CP自身互作的作用位点,分别从CP的N端和C端截短若干个氨基酸序列,结果发现CP的自身互作是高度敏感的,任何较少氨基酸序列的缺失都将导致其自身互作的丧失。本研究为深入探讨病毒的组装机制和致病机理奠定了理论基础。We cloned the genes of infection hypodermal and hematopoietic necrosis virus （IHHNV） of penaeid shrimp into the prey vectors pGADT7 and bait vectors pGBKT7 of Yeast Two-Hybrid System （Matchmaker）. The constructed prey and bait plasmids were co-transformed into yeast strain AH109 to test the self-activation and toxicity. The results show that the recombined prey vectors and bait vectors were not self-activated, and were not toxic to AH109. Subsequently, the recombined prey vectors and bait vectors were co-transformed to AH109 and spread onto SD/-Leu/-Trp mediums, and then spot on SD/-Ade/-His/-Leu/-Trp/X-ct-gal. Phenotypic identification showed that the co-transformation of pGADT7-CP and pGBKT7-CP could grow well and turn blue on plate while all others failed, indicating that only CP could interact with itself. A series of study on truncated CPs indicated that CP-CP interaction was extremely sensitive to any modification of CP. This study may provide a theoretical base for future investigations on mechanisms of virus assembly and pathogenesis.

关 键 词：传染性皮下及造血组织坏死病毒 编码蛋白 相互作用 自身互作 酵母双杂交 

分 类 号：S945.4[农业科学—水产养殖]