香蕉条斑病毒云南分离物ORFⅠ的原核表达及其抗血清制备

Prokaryotic Expression of ORFⅠof Banana Streak Virus Yunnan Isolate and Preparation of a Virus-specific Antiserum

以连接在pMD-18T载体上的香蕉条斑病毒云南分离物(BSV)全基因组为模板,设计特异引物,PCR扩增获得ORFⅠ全长序列。目的片段及原核表达载体pET32(b)分别经EcoRⅠ和XhoⅠ双酶切后,连接构建重组质粒pET32-ORFⅠ,将鉴定为正确的重组质粒转化E.coli BL21(DE3)感受态细胞,获得原核表达OFRⅠ工程菌。工程菌在28℃条件下,用浓度为0.1 mmol/L的IPTG诱导表达6 h,离心收集菌液并超声波破碎,获得以含目的肽段的可溶性蛋白为主的融合蛋白。将融合蛋白通过Ni2+-NTA亲和层析柱纯化后,得到纯度较高的融合蛋白。以此蛋白为抗原免疫家兔,获得特异抗血清,该抗血清的效价达1∶100 000,Western Blot验证表明,抗血清的特异性较强。本研究为BSV的快速检测以及BSV编码蛋白的功能研究奠定了基础。

The ORFⅠsegment was amplified by PCR utilizing the whole genome of BSV Yunnan isolate as the template,then subcloned in the prokaryotic expressional vector pET-32b（＋）simultaneously digested by both EcoRⅠand XhoⅠ.The recombinant plasmid pET32-ORFⅠ was then transformed into the competent cells of E.coli BL21（DE3）.Then the genetic engineering bacterial strain harboring pET32-ORFⅠ was induced by 0.1 mmol/L IPTG at 28 ℃,and the soluble fusion protein including the ORFⅠprotein was generated and then purified by the Ni2＋-NTA column.The purified protein was then utilized to immune a rabbit in order to obtain the virus-specific antiserium.The tilter of the antiserium by indirect enzyme-linked immunosorbent assay（ID-ELISA）was up to 1∶100,000.The antiserium was proved to have high specificity by Western Blot.The present study had paved a way to establish the methods of BSV detection and the research protein function of BSV.