摘要
为了解白斑综合征病毒(white spot syndrome virus,WSSV)两种囊膜蛋白VP281和VP31的功能,本实验对二者进行了原核重组表达。利用一种组成型分泌原核表达质粒pBTA1为表达载体,构建了组成型分泌WSSV囊膜蛋白rVP281、rVP28以及rVP28与增强型绿色荧光蛋白rEGFP融合蛋白的重组大肠杆菌菌株DH5α,分别命名为DhpVP281、DhpVP28和DhpVP28-EGFP。3种重组菌在LB固体培养基上生长12 h的菌落直径分别为(164.84±28.44)、(560.47±46.04)和(548.21±58.54)μm,生长19 h的菌落直径分别为(436.31±47.56)、(1 136.90±110.88)和(1 083.33±109.83)μm,生长24 h的菌落直径分别为(594.19±57.17)、(1 251.19±188.86)和(1 264.29±172.78)μm;显示在所有培养时间,DhpVP281的菌落均显著小于DhpVP28或DhpVP28-EGFP的菌落(P<0.05),推测rVP281的表达可能抑制大肠杆菌的生长。以pBTA1为表达载体,未能成功构建组成型分泌rVP31的重组DH5α。为了解其原因,使用pET-30a(+)为表达载体,构建了表达rVP31包涵体的重组菌株BL21(DE3)pLysS。将rVP31蛋白纯化并复性后,采用牛津杯法,检测到rVP31蛋白对溶壁微球菌具有抗菌作用。在动物病毒中发现具有抗菌作用的囊膜蛋白,可以增加有关WSSV的病毒学方面的知识。
In order to understand the functions of white spot syndrome virus（WSSV） envelope proteins VP281 and VP31,the method of prokaryotic expression was used in this study.By using a prokaryotic constitutive secretory expression plasmid pBTA1 as the expression vector,the recombinant Escherichia coli strain DH5α that could constitutively secrete WSSV envelope protein rVP281,rVP28 or the fusion protein of VP28 and the enhanced green fluorescence protein rEGFP were constructed.They were respectively named as DhpVP281,DhpVP28 and DhpVP28-EGFP.The three recombinant bacteria were cultured on the LB plates in the same condition and their clone diameters were respectively（164.84±28.44） μm,（560.47±46.04） μm and（548.21±58.54） μm when cultured for 12 hours,（436.31±47.56） μm,（1136.90±110.88） μm and（1083.33±109.83） μm when cultured for 19 hours and（594.19±57.17） μm,（1251.19±188.86） μm and（1264.29±172.78） μm when cultured for 24 hours.It was displayed that the clones of DhpVP281 were significantly smaller than those of DhpVP28 and DhpVP28-EGFP in the whole culturing period（P0.05） and we guessed that the expression of rVP281 probably inhibited the growth of E.coli.However,recombinant bacteria constitutively expressing VP31 can not be constructed with the vector pBTA1.In order to find out the reason,recombinant bacteria BL21（DE3） pLysS were constructed with pET-30a（＋） as the expression vector and rVP31 was obtained in the form of inclusion body.After renaturation,the antibacterial activity of rVP31 was tested.The result showed that rVP31 has the antibacterial activity against Micrococcus lysodeikticus.These findings,reported for the first time,may increase the virology knowledge of WSSV.
出处
《水产学报》
CAS
CSCD
北大核心
2012年第12期1901-1909,共9页
Journal of Fisheries of China
基金
国家重点基础研究项目(2006CB101804)
关键词
白斑综合征病毒
囊膜蛋白
抗菌活性
white spot syndrome virus（WSSV）
envelope proteins
antibacterial activity
