猪流行性腹泻病毒COE蛋白的原核表达及免疫原性分析  

Prokaryotic Expression and Immunogenicity Analysis of COE Protein of Porcine Epidemic Diarrhea Virus

作　　者：李雅心 郭涛 胡瑞瑞 王小奎[1] 米桃桃 倪伟[1] 胡圣伟[1] LI Ya-xin;GUO Tao;HU Rui-rui;WANG Xiao-kui;MI Tao-tao;NI Wei;HU Sheng-wei(College of Life Sciences,Shihezi University,Shihezi,Xinjiang,832000,China)

机构地区：[1]石河子大学生命科学学院,新疆石河子832000

出　　处：《动物医学进展》2022年第3期1-5,共5页Progress In Veterinary Medicine

基　　金：2019年新疆维吾尔自治区研究生科研创新项目(XJ2019G11);新疆“兵团英才”第二周期第一层次培养人选项目。

摘　　要：为表达猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)中和抗原表位COE蛋白,分析其免疫原性,为PEDV的检测和亚单位疫苗的开发奠定基础。参考GenBank中PEDV COE基因序列,人工合成并经生物信息学综合分析后,将合成的基因片段插入原核表达载体pET-32a(+),构建重组表达质粒pET-32a-COE,将鉴定正确的重组质粒转化至大肠埃希氏菌TransB(DE3)中,用IPTG诱导目的蛋白表达,并进行SDS-PAGE鉴定;然后用纯化的目的蛋白与弗氏不完全佐剂乳化,免疫猪后用ELISA方法检测猪血清中PEDV特异性抗体水平。结果表明,目的蛋白在大肠埃希氏菌TransB(DE3)中获得表达,用His标签纯化得到了与预期大小35 ku相符的COE融合蛋白,经复性、浓缩后的蛋白浓度为1.85 mg/mL。用该蛋白作为亚单位疫苗免疫猪,ELISA方法检测免疫猪产生了高滴度的血清PEDV特异性抗体。表明原核表达的COE蛋白具有良好的免疫原性,可作为候选的亚单位疫苗。Prokaryotic expression of porcine epidemic diarrhea virus neutralizing epitope COE protein to detect its immunogenicity will lay the foundation for the detection of PEDV and the development of subunit vaccines.The gene of PEDV COE was synthesized and analyzed by bioinformatics.The gene was inserted into the prokaryotic expression vector pET-32a(+)to construct the recombinant expression plasmid pET-32a-COE.The recombinant plasmid was identified by EcoRⅠand XhoⅠdigestion.The recombinant plasmid was transformed into E.coli TransB(DE3).The expression of the target protein was induced by IPTG and identified by SDS-PAGE.The successfully expressed target protein was purified and emulsified with Freund's incomplete adjuvant.After the pigs were immunized,the changes in serum PEDV specific antibodies were detected by ELISA.The pET-32a-COE expression vector was constructed,and the COE fusion protein was successfully expressed in E.coli TransB(DE3).The COE fusion protein was purified by His tag and the expected size of 35 kDa.The protein concentration after renaturation is 1.85 mg/mL.After the protein was used as a subunit vaccine to immunize pigs,high titers of serum PEDV-specific antibodies were produced.The COE protein expressed in prokaryotic cells can induce the body to produce high-titer PEDV-specific antibodies with good immunogenicity.

关 键 词：猪流行性腹泻病毒 中和抗原表位 COE基因 原核表达 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]