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当前位置: 首页 > 文章 > 猪流行性腹泻病毒COE蛋白原核表达及其反应原性的初步鉴定 黑龙江畜牧兽医 2016 (23) 152-155
Position: Home > Articles > Prokaryotic expression of the COE protein of porcine epidemic diarrhea virus and preliminary identification of its reactionogenicity Heilongjiang Animal Science and Veterinary Medicine 2016 (23) 152-155

猪流行性腹泻病毒COE蛋白原核表达及其反应原性的初步鉴定

作  者：
孙彦阔;汪志;冯嘉萍;石庆伟;罗勇峰
单  位：
华南农业大学兽医学院;华南农业大学兽医学院国家生猪种业工程技术研究中心
关键词：
猪流行性腹泻病毒(PEDV);S基因;逆转录PCR;原核表达;可溶性COE蛋白;免疫印迹反应
摘  要：
为了研究猪流行性腹泻病毒(PEDV)的优势抗原表位COE可溶性原核表达及其反应原性,试验采用RT-PCR方法扩增出COE基因,将其克隆至原核表达载体p MAL-C5X上,构建重组质粒p MAL-C5X-COE,经测序鉴定正确后转化Rosetta感受态细胞,并进行IPTG诱导表达和Westernblot验证。结果表明:重组菌p MAL-C5X-COE表达的融合蛋白MBP-COE的分子质量约为57 ku,优化的诱导条件为IPTG浓度0.8 mmol/L、诱导时间4 h,诱导温度30℃,重组蛋白以可溶性蛋白形式存在于菌体中,融合蛋白与兔抗PEDV多克隆抗血清发生特异的免疫印迹反应。说明原核表达的COE融合蛋白可溶且具有良好的反应原性。
译  名：
Prokaryotic expression of the COE protein of porcine epidemic diarrhea virus and preliminary identification of its reactionogenicity
作  者：
SUN Yankuo;WANG Zhi;FENG Jiaping;SHI Qingwei;LUO Yongfeng;QIN Feng;BAI Xiaolei;ZHANG Guihong;College of Veterinary Medicine,South China Agricultural University;National Engineering Research Center For Beeding Swine Industry;
关键词：
Porcine epidemic diarrhea virus(PEDV);;S gene;;reverse transcription PCR(RT-PCR);;prokaryotic expression;;soluble COE protein;;Western blot reaction
摘  要：
To study the soluble prokaryotic expression and reactogenicity of the core neutralizing epitope( COE) of porcine epidemic diarrhea virus( PEDV),RT- PCR assay was used to amplify the COE gene. The COE gene was cloned into a prokaryotic expression vector p MAL- C5 X to construct a recombinant plasmid p MAL- C5X- COE. After sequencing,the recombinant plasmid was transformed into Escherichia Rosetta competent cells and induced by IPTG. Then the expression was verified through Western- blot. The result showed that the molecular mass of the fusion protein MBP- COE expressed by recombinant strain p MAL- C5X- COE was about 57 ku. Optimized conditions were as follows:IPTG concentration of 0. 8 mmol/L,induction time of 4 h,and induction temperature of 30 ℃. The recombinant protein existed in the bacteria with a soluble form of protein,and the fusion protein had specific Western blot reaction with anti PEDV rabbit polyclonal antiserum. The result indicates that the prokaryotic expressed COE fusion protein is soluble and has good reactogenicity.

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