摘要
克隆并原核表达新疆株猪圆环病毒3型(PCV3)衣壳蛋白(Cap)基因,为PCV3检测试剂的研发奠定基础。采用PCR扩增PCV3新疆分离株的Cap基因,采用Chou-Fasman法、Karplus-Schulz法、Kyte-Doolittle法、Emini法和Jameson-Wolf法预测蛋白的二级结构、蛋白质骨架区的柔韧性、亲水区/疏水区、可及性和抗原指数,将PCV3 Cap基因克隆到pEASY-Blunt Simple载体,经HindⅢ、NdeⅠ双酶切,亚克隆至原核表达载体pET-30a(+),构建重组质粒pET30a-PCV3-Cap,测序验证后转入大肠埃希氏菌BL21(DE3)感受态细胞,通过IPTG诱导表达,采用SDS-PAGE和Western blot对表达的重组蛋白进行分析鉴定。结果显示,成功扩增并克隆到新疆株PCV3 Cap基因,通过序列分析发现所获PCV3 Cap基因与参考毒株的同源性为98.0%~98.6%之间,推导氨基酸有6个点突变,分别位于24、27、29、56、75、77和150aa位置;在11-17、37-41、54-60、97-101、119-134、138-140、142-146、155-160、170-172、175-181和192-202aa含有潜在B细胞抗原表位。构建了重组原核表达质粒pET30a-PCV3-Cap,并在大肠埃希氏菌中表达了重组PCV3 Cap蛋白,分子质量约为32 ku,该重组蛋白以包涵体的形式存在,Western blot鉴定表明,带His标签的重组蛋白能被His单克隆抗体识别。成功克隆并原核表达了新疆株PCV3衣壳蛋白,为PCV3诊断试剂的研发奠定了基础。
In order to clone and prokaryotically express the capsid protein(Cap)gene of Xinjiang porcine circovirus type 3(PCV3),and lay a foundation for the development of diagnostic reagents for PCV3.The PCR method was used to amplify the Cap gene.The Chou-Fasman method,the Karplus-Schulz method,the Kyte-Doolittle method,the Emini method and the Jameson-Wolf method were used to predict the secondary structure of the protein,the flexibility of the protein skeleton region,and the hydrophilic region/hydrophobic region,accessibility and antigenic index,PCV3 Cap gene was cloned into pEASY-Blunt Simple vector,digested with HindⅢand NdeⅠ,subcloned into prokaryotic expression vector pET-30a(+),and recombinant plasmid pET30a-PCV3-Cap was constructed.PCV3-Cap gene was sequenced and transformed into BL21(DE3)competent cells,which were induced by IPTG.The expressed recombinant proteins were identified by SDS-PAGE and Western blot.The results showed that PCV3 Cap gene was successfully amplified.The sequence analysis showed that the homology between the PCV3 Cap gene of Xinjiang strain and the reference strains was between 98.0%and 98.6%,and the deduced amino acid had 6 point mutations,respectively in the 27,29,56,75,77,and 150 aa positions;at 11-17,37-41,54-60,97-101,119-134,138-140,142-146,155-160,170-172,175-181 and 192-202aa,there were contain potential B cell epitopes.The recombinant prokaryotic expression plasmid pET30a-PCV3-Cap was successfully constructed and the recombinant PCV3 Cap protein was expressed in E.coli.The molecular weight was about 32 ku.The recombinant protein existed in the form of inclusion bodies.Western blot analysis showed that the recombinant protein with His tag could be recognized by his monoclonal antibody.This study successfully cloned and prokaryotically expressed the PCV3 capsid protein of Xinjiang strain,which laid the foundation for the development of PCV3 diagnostic reagent.
作者
任敏
屈勇刚
魏其
谷思颖
杨慧敏
吴圆圆
于会举
REN Min;QU Yong-gang;WEI Qi;GU Si-ying;YANG Hui-min;WU Yuan-yuan;YU Hui-ju(College of Animal Science and Technology,Shihezi Univerisity,Shihezi,Xinjiang,832003,China;Department of Bioengineering,BaYin GuoLeng Vocational and Technical College,Korla,Xinjiang,841002,China)
出处
《动物医学进展》
北大核心
2021年第1期18-24,共7页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(U1503283)。
关键词
猪圆环病毒3型
衣壳蛋白
原核表达
Porcine circovirus type 3
capsid protein
prokaryotic expression
