| 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(Infectionhypodermal 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pGADT7和诱饵载体pGBKT7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pGADT7-CP/pGBKT7-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。 |
| 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 |
| DOI：10.11693/hyhz20150900233 |
| 分类号: |
| 基金项目: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。 |
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| INTERACTIONS BETWEEN INFECTIOUS HYPODERMAL AND HEMATOPOIETIC NECROSIS VIRUS (IHHNV) ENCODING PROTEINS OF PENAEID SHRIMP |
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CHEN Shen-Xue,WEI Yong-Wei,YANG Zhi-Jing,MIAO Liang and CHEN Jiong
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Laboratory of Biochemistry and Molecular Biology,School of Marine Sciences,Ningbo University,Laboratory of Biochemistry and Molecular Biology,School of Marine Sciences,Ningbo University,Laboratory of Biochemistry and Molecular Biology,School of Marine Sciences,Ningbo University,Laboratory of Biochemistry and Molecular Biology,School of Marine Sciences,Ningbo University,Laboratory of Biochemistry and Molecular Biology,School of Marine Sciences,Ningbo University
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| Abstract: |
| 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-α-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. |
| Key words: Infectious hypodermal and haematopoietic necrosis virus (IHHNV) encoding proteins interaction self-interaction yeast two-hybrid |
