博尔纳病病毒磷蛋白的原核表达及纯化

Prokaryotic Expression and Purification of Borna Disease Virus Phosphoprotein

目的构建博尔纳病病毒(Borna disease virus,BDV)磷蛋白(p24蛋白)基因重组表达质粒,原核表达p24蛋白,并进行纯化。方法通过RT-PCR法从BDV感染的少突胶质细胞株(Oligodendrocyte,OL)中扩增p24基因片段,构建重组表达质粒pET-41a-p24,转化大肠杆菌BL21(DE3),IPTG诱导表达,并对表达的重组蛋白进行亲和层析纯化。结果经双酶切鉴定和测序证实,重组表达质粒pET-41a-p24构建正确;表达的重组p24蛋白相对分子质量约24 000,表达量占菌体总蛋白的30%以上,主要以可溶形式表达;纯化的重组蛋白纯度达85%左右,可与鼠抗BDV p24单克隆抗体特异性结合。结论已成功原核表达并纯化了重组BDV p24蛋白,为进一步建立BDV相关免疫学检测方法奠定了基础。

Objective To construct the recombinant plasmid carrying the gene encoding Borna disease virus（BDV） phosphoprotein（p24）,express in prokaryotic cells and purify the expressed product.Methods The p24 gene was amplified by RT-PCR from oligodendrocytes（OL） infected with BDV and cloned into prokaryotic expression vector pET-41a（＋）.The constructed recombinant plasmid pET-41a-p24 was transformed to E.coli BL21（DE3） for expression under induction of IPTG.The expressed protein was purified by affinity chromatography.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-41a-p24 was constructed correctly.The expressed p24,with a relative molecular mass of about 24 000,contained more than 30% of total somatic protein and mainly existed in a soluble form.The purified recombinant protein reached a purity of about 85% and showed specific binding to mouse anti-BDV p24 McAb.Conclusion Recombinant BDV p24 protein was successfully expressed in prokaryotic cells,which laid a foundation of further development of the relevant methods for immunoassay.