4种分子伴侣促进猫细小病毒样颗粒可溶性表达的研究

Study on four molecular chaperones promoting the soluble expression of virus-like particles of Feline parvovirus

为了研究4种分子伴侣在E.coli表达系统中对猫细小病毒(feline Parvovirus, FPV)VP2蛋白表达及其组装的病毒样颗粒(virus-like particles, VLPs)活性的影响,试验构建重组质粒pET30a-VP2,将重组质粒pET30a-VP2分别与4种分子伴侣pKJE7、pKJE8、pGro7和pTf16转化至E.coli ER2566感受态细胞中进行共表达,运用SDS-PAGE、Western-blot对表达产物进行检测,通过硫酸铵沉淀结合疏水层析柱对表达产物进行纯化,电镜观察VLPs的形态,并采用血凝试验检测其免疫活性。结果表明:重组质粒pET30a-VP2在E.coli表达系统中主要以包涵体形式表达蛋白。4种分子伴侣与重组质粒pET30a-VP2共表达,加入终浓度为2 mg/mL L-阿拉伯糖及0.1 mmol/L IPTG或5 ng/mL四环素的诱导剂,25℃诱导16 h,均能显著提高FPV VP2蛋白的可溶性表达,表达蛋白具有抗原特异性。纯化得到的FPV VP2蛋白在250 mmol/L NaCl(pH值为8.0)条件下可自组装形成均一、直径约为24 nm的VLPs。仅分子伴侣pTf16与重组质粒pET30a-VP2共表达获得的FPV VP2蛋白经透析形成的FPV VLPs正常,无碎片,其血凝效价为219。说明通过将重组质粒pET30a-VP2分别与4种分子伴侣在E.coli表达系统中共表达,4种分子伴侣均可促进FPV VP2蛋白的可溶性表达,成功纯化并获得FPV VLPs。

In order to study the effect of four types of molecular chaperones in the expression of Feline parvovirus(FPV) VP2 in soluble form in E. coli,and in the activity of the assembled virus-like particles(VLPs),the recombinant plasmid pET30 a-VP2 was constructed in this study, which was transformed with four types of molecular chaperones pKJE7,pKJE8,pGro7 and pTf16 respectively into E. coli ER2566 competent cells for co-expression. SDS-PAGE and Western-blot were adopted in the detection of expressed products which were purified by ammonium sulfate precipitation combined with hydrophobic chromatography. The morphology of VLPs were analyzed by electron microscopy observation, and their immunocompetence was identified by hemagglutination test. The results showed that the recombinant plasmid pET30 a-VP2 expressed in E. coli obtained the protein mainly in inclusion body form. Four types of molecular chaperones with the recombinant plasmid pET30 a-VP2 were carried out for co-expression. The inductive agents were the final concentrations of 2 mg/mL L-Arabinose and 0.1 mmol/L IPTG or 5 ng/mL tetracycline, 25 ℃ for 16 h, which could significantly increase the soluble expression of FPV VP2 protein;and the expressed protein had good antigen specificity. The purified FPV VP2 protein could self-assemble to form uniform VLPs with a diameter of about 24 nm under the condition of 250 mmol/L NaCl(pH 8.0). The FPV VP2 protein co-expressed by the molecular chaperone pTf16 and the recombinant plasmid pET30 a-VP2 was dialyzed to form FPV VLPs, which had hemagglutination titer 219. The results suggested that by co-expressing the recombinant plasmid pET30 a-VP2 with four molecular chaperones in a prokaryotic expression system, all four chaperones can promote the soluble expression of FPV VP2 protein, and FPV VLPs were successfully purified and obtained.