羊口疮病毒安徽株116基因的生物信息学分析及原核表达
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国家重点研发计划(2018YFD0502006), 2019年国家级大学生创新训练计划(201910364044)和安徽省大学生创新训练计划(201810364023)共同资助。


Bioinformatics analysis and prokaryotic expression of Orf virus Anhui strain 116 gene
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    摘要:

    为了对羊口疮病毒(Orf virus, ORFV)安徽株116基因进行生物信息学分析及原核表达。以ORFVAH-F10株为模板PCR扩增116基因,使用生物信息学软件预测该基因编码蛋白的结构与功能。同时将扩增产物克隆至pET-32a(+)原核表达载体,经BamH I及EcoR I双酶切鉴定后进行测序,构建pET-32a(+)-116重组质粒。转化至大肠杆菌Rosetta(DE3)进行IPTG诱导,纯化蛋白后免疫BALB/c雌性小鼠制备多克隆抗体。测序结果显示,ORFV116基因全长561 bp,编码186个氨基酸。生物信息学分析结果显示:该蛋白分子量约为20.33 kDa,为不稳定亲水性蛋白;无信号肽、保守结构域及跨膜结构域;含有2个N糖基化位点和42个磷酸化位点;二级结构以无规则卷曲为主,分别为延伸链(5.91%)、α-螺旋(14.52%)与无规则卷曲(79.57%);预测该蛋白含有20个可能的B细胞优势抗原表位与4个CTL细胞表位。SDS-PAGE及Western-blot结果显示,ORFV116蛋白大小约为51 kDa,主要以可溶形式表达且具有良好的反应原性。

    Abstract:

    The study was aimed at analyzing the bioinformatics and prokaryotic expressing the 116 gene of Anhui Orf virus (ORFV). The 116 gene was amplified by PCR, with the ORFV AH-10 strain as the template, and the structural and function of the coding protein were predicted by the bioinformatics technique. The amplification product was cloned into a prokaryotic vector pET-32a(+), and sequenced after double enzyme digestion identification by BamH I and EcoR I. The pET-32a(+) -116 was constructed and transformed to E. coli Rosetta (DE3) for IPTG induction. The induction conditions were optimized and the purified protein was obtained. The purified ORFV116 was used as an immunogen to inject into BALB/c mouse to produce anti-ORFV116 polyclonal antibody (pAb). The results showed that the 116 gene of ORFV, 561 bp, encoded 186 amino acids. Bioinformatics analysis showed that the encoded protein was an unstable hydrophilic protein with a relative molecular weight of 20.33 kDa. It contained two N glycosylation sites and forty-two phosphorylation sites, with no signal peptide, conserved domain and transmembrane domain. In the secondary structure, the random coil accounted for 79.57%, the extended chain and α-helix accounted for 5.91% and 14.52%, respectively. Twenty potential dominant epitopes of B cells and four CTL epitopes were predicted and analyzed. The SDS-PAGE and Western-blot results showed that ORFV116 protein was approximately 51 kDa , which was mainly expressed in soluble form with good reactogenicity.

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  • 在线发布日期: 2021-01-22