摘要
为了对羊口疮病毒(ORFV)安徽株121基因进行原核表达及生物信息学分析。以ORFV AH-F10株为模板对121基因进行PCR扩增,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-1-121,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,并对重组蛋白的诱导条件进行优化,确定了ORFV 121重组蛋白表达的最佳条件。SDS-PAGE及Western-blot鉴定结果表明,重组蛋白大小约为60 ku,在Rosetta中高效表达,主要以包涵体形式存在且具有良好的反应原性。包涵体蛋白经过纯化后得到目的蛋白,将纯化蛋白与弗氏佐剂进行乳化后皮下注射6周龄的BALB/c雌鼠。每14 d后加强免疫1次,4次后收集小鼠血清。对收集到的血清进行Western-blot特异性鉴定,该抗体血清可以和阳性原核表达的ORFV 121蛋白进行特异性反应,表明成功制备鼠源多抗血清。利用生物信息学相关软件对目的基因编码蛋白的理化性质、信号肽、跨膜结构域、磷酸化位点、二级结构和B细胞表位进行预测。结果显示该蛋白等电点为8.86,属不稳定的亲水性蛋白;预测有50个可能的磷酸化位点,无信号肽和跨膜结构域;二级结构中以无规则卷曲结构居多占62.25%,α-螺旋、β-转角、延伸链分别占27.81%,2.98%,6.95%;预测该蛋白存在8个潜在B细胞优势表位。成功表达了ORFV AH-F10121蛋白并预测其生物学特性,为该蛋白结构与功能及ORFV致病机理的深入研究奠定基础。
In order to clone and express 121 gene of ORFV Anhui strain and analyze the gene bioinformatics.In this study,the 121 gene was PCR amplified,with the ORFV AH-F10 strain as the template.The amplified product was cloned into pGEX-6P-1 prokaryotic expression vector,and the recombinant plasmid pGEX-6P-1-121 was constructed and identified by sequencing.Then,the correctly identified recombinant plasmid was transformed into E.coli Rosetta competent cells,and was induced by IPTG.And optimizing the induction conditions of the recombinant protein to determine the optimal conditions for the expression of ORFV 121 recombinant protein.The results of SDS-PAGE and Western-blot showed that the size of the recombinant protein was about 60 ku,which was highly expressed in Rosetta and mainly in the form of inclusion body with good reaction genicity.After inclusion body protein was purified to obtain the target protein,emulsified with Freund′s adjuvant to inject subcutaneously into BALB/c female mice aged 6 weeks.The mice were booster immunized once every 14 d,and serum was collected after 4 times,which was specifically identified by Western-blot.The results showed the antibody serum could specifically react with ORFV 121 protein of positive prokaryotic expression,indicating the successful preparation of multi-antiserum.In addition,the physicochemical properties,signal peptides,transmembrane domains,phosphorylation sites,secondary structures and B-cell epitopes of the target gene coding proteins were predicted by bioinformatics software.The results of bioinformatics indicated that the isoelectric point of the protein was 8.86,which was an unstable hydrophilic protein.It was predicted that there were 50 possible phosphorylation sites,no signal peptide and transmembrane domain.In the secondary structure,the random coil accounted for 62.25%,theα-helix,β-turn and extended chain accounted for 27.81%,2.98%and 6.95%,respectively.Eight potential dominant epitopes of B cells were predicted and analyzed.Inconclusion,the ORFV 121 protein o
作者
刘自敏
王小朋
白彩霞
杨侃侃
张学琪
胡子慧
孙裴
王勇
LIU Zimin;WANG Xiaopeng;BAI Caixia;YANG Kankan;ZHANG Xueqi;HU Zihui;SUN Pei;WANG Yong(College of Animal Science and Technology, Anhui Agricultural University, Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control,Hefei 230036, China)
出处
《华北农学报》
CSCD
北大核心
2019年第S01期358-365,共8页
Acta Agriculturae Boreali-Sinica
基金
国家重点研发计划(2018YFD0502006)
国家自然科学基金项目(31602063)
安徽农业大学大学生科技创新基金项目
