| 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。 |
| 关键词: WSSV VP110 原核表达 凡纳滨对虾鳃细胞膜蛋白 Far-western |
| DOI：10.11758/yykxjz.20140210 |
| 分类号: |
| 基金项目:公益性行业（农业）科研专项经费（201103034）、现代农业产业技术体系（CARS-47）和中国水产科学研究院黄海水产研究所级基本科研业务费（20603022013009）、“泰山学者”建设工程专项经费和农业科研杰出人才培养计划项目 |
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| Partial expression of white spot syndrome virus (WSSV) envelope protein VP110 and its binding activity with cellular membrane proteins of shrimp gills |
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| Abstract: |
| 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. |
| Key words: WSSV VP110 Prokaryotic expression Cellular proteins of Litopenaeus vannamei gills Far-Western |
