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

中文关键词: WSSV  VP110  原核表达  凡纳滨对虾鳃细胞膜蛋白  Far-western

英文关键词: WSSV  VP110  Prokaryotic expression  Cellular proteins of Litopenaeus vannamei gills  Far-Western

中文摘要:

      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。

英文摘要:

      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 (LT50) 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 MALDI-TOFMS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) proved that this 41 kDa protein is actin.