文章摘要
张正阳,张春莉,施定基,贾晓会,贾睿,何培民.白斑综合征病毒(WSSV)基因VP28原核表达与检测[J].上海海洋大学学报,2017,26(2):183-188.
ZHANG Zhengyang,ZHANG Chunli,SHI Dingji,JIA Xiaohui,JIA Rui,HE Peimin.Prokaryotic gene expression and detection of white spot syndrome virus (WSSV)VP28[J].Journal of Shanghai Ocean University,2017,26(2):183-188.
白斑综合征病毒(WSSV)基因VP28原核表达与检测
Prokaryotic gene expression and detection of white spot syndrome virus (WSSV)VP28
投稿时间:2016-04-14  修订日期:2016-12-02
DOI:10.12024/jsou.20160401739
中文关键词: 对虾  白斑综合征病毒(WSSV)  VP28  大肠杆菌  鱼腥藻7120  定量免疫印迹
英文关键词: shrimp  white spot syndrome virus(WSSV)  VP28  Escherichia coli  Anabaena sp. PCC 7120  quantitative Western blotting
基金项目:国家高技术研究发展计划(2014AA093506);上海市科委项目(16391903500)
作者单位E-mail
张正阳 上海海洋大学 水产与生命学院, 上海 201306  
张春莉 Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany  
施定基 中国科学院植物研究所, 北京 100093  
贾晓会 上海海洋大学 水产与生命学院, 上海 201306  
贾睿 上海海洋大学 水产与生命学院, 上海 201306  
何培民 上海海洋大学 水产与生命学院, 上海 201306 pmhe@shou.edu.cn 
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中文摘要:
      VP28是对虾白斑综合征病毒(White Spot Syndrome Virus,WSSV)的囊膜蛋白。将VP28基因序列密码子优化后合成,经限制性内切酶Nde Ⅰ、Xho Ⅰ酶切后,按正确的阅读框顺序插入到pET-28a(+)表达载体上。重组质粒转化大肠杆菌BL21(DE3),经质粒双酶切、测序鉴定后成功地构建了VP28基因原核表达载体pET-28a-VP28。转化菌经IPTG诱导,SDS-PAGE显示含有与预期大小一致的约30 ku蛋白带,主要以包涵体形式表达。采用Ni-IDA-Sepharose CL-6B亲和层析柱对重组蛋白进行纯化,获得了纯度为95%的大肠杆菌重组蛋白VP28。该纯化蛋白作为绝对定量Western的标准品,梯度稀释建立标准曲线,以定量检测转基因鱼腥藻7120中的VP28绝对表达量。实验结果表明,大肠杆菌BL21(DE3)表达的重组蛋白VP28与鱼腥藻7120中表达的VP28分子量基本相同,纯化后的大肠杆菌重组蛋白梯度稀释作为绝对定量Western标准曲线,计算出转基因鱼腥藻7120在培养第17天时,VP28的表达量最大,为5.14 μg/mL,占转基因鱼腥藻7120总蛋白浓度的1.45%。这不仅对转基因鱼腥藻的高效培养具有重要意义,也为日后确定投喂对虾口服疫苗有效剂量防治白斑综合征奠定了基础。
英文摘要:
      VP28 is the envelope protein of white spot syndrome virus (WSSV).After the codon optimization synthesis VP28 gene sequence,and enzyme digestion by restriction enzymes Nde I, Xho I, inserted the correct reading frame sequence into the expression vector-pET-28a(+).After the recombinant plasmid into E. coli BL21 (DE3) was digested by double enzyme and sequencing, it was confirmed that we have successfully constructed VP28 genetic prokaryotic expression vector-pET-28a-VP28.The transferred strain induced by IPTG, SDS-PAGE showed that contains the expected size of about 30 ku protein band, which was mainly expressed in inclusion body form. The recombinant protein was purified by Ni-IDA-Sepharose CL-6B affinity chromatography column and we got the purity of 95% protein finally.Gradiently dilute the purified protein as the quantitative standard curve of Western blotting, to quantify the amount of VP28 expression in transgenic Anabaena sp. PCC 7120.The experiment results showed that the recombinant protein and VP28 expressed in Anabaena sp. PCC 7120 had the same molecular weight.Use the purification of recombinant protein as a quantitative western blotting standard curve, calculate the expression of VP28 in transgenic Anabaena sp. PCC 7120.The largest amount was 5.14 μg/mL, on 17th day, accounting for 1.45% of the total protein concentration.This is not only of great significance to efficient cultivation of transgenic cyanobacteria, but also a foundation to determine the effective dosage of oral vaccine for prawn to prevent and cure white spot syndrome in the future.
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