对虾白斑综合征病毒(WSSV)囊膜蛋白VP110部分重组表达及其与对虾鳃细胞膜蛋白结合分析

Partial expression of white spot syndrome virus(WSSV) envelope protein VP110 and its binding activity with cellular membrane proteins of shrimp gills

将VP110基因的部分序列克隆到pET-28a载体中构建pET28a-vp110b重组质粒并进行原核表达,获得重组表达的蛋白rVP110-B;用rVP110-B注射凡纳滨对虾Litopenaeus vannamei后,经WSSV感染,实验表明,该蛋白注射使凡纳滨对虾感染WSSV的半数死亡时间比对照组延长了20%。用表达纯化的该重组蛋白制备了兔抗rVP110-B多克隆抗体,该抗体用于凡纳滨对虾鳃细胞膜蛋白与rVP110-B的Far-western分析显示,凡纳滨对虾鳃细胞膜蛋白中除90 kDa左右的血蓝蛋白外,在41.7 kDa存在结合条带,经质谱分析表明这条鳃细胞膜蛋白是肌动蛋白。

VP110,an envelope protein of white spot syndrome virus （WSSV）,with a molecular weight of 110 kDa,has an Arg-Gly-Asp （RGD） structure domain and the ability of binding gill cells of the host.In order to study the role of VP110 in the WSSV infection of Litopenaeus vannamei,we designed a pair of primers according to the partial sequence of the gene vp110 （vp110-b）.The vp110-b was then amplified by PCR and cloned into Escherichia coli expression vector pET-28a successfully.The pET28a-vp110b was transformed into E.coli BL21 cells,and a fusion protein rVP110-B （50 kDa） was expressed.The rVP110-B was injected into L.vannamei,and the protecting effect against WSSV infection was evaluated.The results showed that the half lethal time （LTs0） of L.vannamei treated with rVP110-B was prolonged by 20% compared to the control.Rabbit anti-VP110 polyclonal antibody,which was prepared with the expressed and purified rVP110-B,was used in the Far-Western analysis of gill cell membrane protein of L.vannamei and renatured rVP110-B.The Far-Western showed a protein band at MW of 41 kDa besides the band of hemocyanin at 90 kDa.Analysis of protein band by MALDITOFMS （matrix-assisted laser desorption ionization-time of flight mass spectrometry） proved that this 41 kDa protein is actin.