摘要
利用PCR方法扩增猪细小病毒7型(porcine parvovirus 7,PPV7)的Cap基因,将扩增产物克隆至pGEX-6P-1原核表达载体,构建重组质粒pGEX-6P-1-Cap,并进行测序鉴定。经鉴定正确后的重组质粒转化至E.coli Rosetta感受态细胞进行IPTG诱导表达,利用SDS-PAGE和Western-blot进行鉴定。同时使用生物信息学软件预测该基因编码的蛋白的理化性质、二级结构、信号肽和跨膜结构域等。结果表明,成功从病料中扩增到Cap基因并构建获得重组质粒pGEX-6P-1-Cap,诱导表达出大小约为78 ku的Cap蛋白,与预期相符。诱导获得的Cap蛋白能够与GST单抗发生特异性反应,具有良好的反应原性。生物信息学分析结果显示,该蛋白是亲水稳定蛋白,二级结构中以无规则卷曲(c)结构居多,占62.90%,α螺旋(h)、β转角(t)、延伸链(e)分别占11.09%、4.05%、21.96%,无信号肽区域和跨膜结构域,存在62个磷酸化位点,12个B细胞抗原表位,2个CTL表位和3个Th表位。本研究成功表达了PPV7 Cap蛋白并预测其生物学特性,为该蛋白质的生物学功能相关研究提供参考。
The porcine parvovirus 7(PPV7)Cap gene was amplified by PCR,and the amplified product was cloned into the prokaryotic expression vector pGEX-6P-1.The recombinant plasmid pGEX-6P-1-Cap was identified by sequencing and the recombinant plasmid was transformed into E.coli Rosetta cells and induced by IPTG.The expression products were then identified by SDS-PAGE and Western-blot.At the same time,physical and chemical properties,secondary structure,signal peptide and transmembrane domain of the gene encoded protein were predicted using the bioinformatics softwares.The results showed that the recombinant plasmid pGEX-6P-1-Cap was successfully constructed and the expressed Cap protein was about 78 ku,which was consistent with the expectation.The induced Cap protein could react specifically with GST monoclonal antibody and had good reactivity.Bioinformatics analysis results showed that the Cap protein was stable hydrophilic protein,with 62.90%irregular curl(c)structure in the secondary structure,11.09%,4.05% and 21.96% of α-helix(h),β-turn(t)and extended(e)chain respectively.Moreover,there was no signal peptide area and transmembrane domain.There were 62 phosphorylation sites,12 B cell antigen epitopes,2 CTL epitopes and 3 Th epitopes.In this study,PPV7 Cap protein was successfully expressed and its biological characteristics were predicted,which could provide a reference for the study of biological function of PPV7 Cap protein.
作者
王小朋
赵靓
刘自敏
白彩霞
杨侃侃
张达
孙裴
蒋书东
李永东
王勇
WANG Xiaopeng;ZHAO Liang;LIU Zimin;BAI Caixia;YANG Kankan;ZHANG Da;SUN Pei;JIANG Shu-dong;LI Yongdong;WANG Yong(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;Ningbo Municipal Center for Disease Control and Prevention,Ningbo 315010,China)
出处
《浙江农业学报》
CSCD
北大核心
2020年第2期200-209,共10页
Acta Agriculturae Zhejiangensis
基金
国家重点研发计划(2018YFD0502006)
国家自然科学基金(31602063)
