摘要
目的表达Ⅱ型副流感病毒核衣壳蛋白,为制备抗体作前期工作。方法从分离培养的Ⅱ型副流感病毒标本中RT-PCR扩增出核衣壳蛋白基因,NdeⅠ和HindⅢ双酶切PCR产物,定向连接到原核表达载体pQE2中,得到重组表达质粒pQEHpI2NP,重组表达质粒进行DNA测序鉴定,转化大肠杆菌BL21(DE3)。IPTG诱导表达,SDS-PAGE检测目的蛋白的表达。利用Ni-NTAagarose纯化带6个组氨酸标记的重组蛋白,重组蛋白免疫小鼠,制备抗HpI2NP多抗,Hep-2细胞培养的Ⅱ型副流感病毒制备可溶性抗原。Western-blot分析。结果构建了表达重组质粒pQE2_HpI2NP,测序鉴定了克隆序列的准确性,在大肠杆菌中成功地表达并纯化得到重组核衣壳蛋白。经Western-blot证实,重组蛋白免疫的小鼠血清与细胞培养的Ⅱ型副流感病毒核衣壳蛋白有特异性反应。结论成功表达了Ⅱ型副流感病毒核衣壳蛋白,并制备了抗HpI2NP多抗。
Objective To construct expression recombinant plasmid containing the nuclear protein gene of human parainfluenza virus type 2 （HpI2） for the production of anti-parainfluenza vaccine. Method The gene for the nuclear protein （NP） of HpI2 was amplified by PCR from cell culture infected with HpI2. The PCR product was cloned into Nde Ⅰ and Hind Ⅲ cut pQE2 vector to yield the recombinant plasmid pQE2-HpI2NP. The cloned fragment was sequenced and the fusion protein was expressed in E.coli BL21. Result The expressing recombinant plasmid pQE2-HpI2NP was constructed. The DNA sequence of the cloned HpI2NP gene was identical to the published sequence in GenBank. The fusion protein was expressed in E.coli and used to produce polyclonal antibodies. The specificity of the anti-NP antibodies was demonstrated by Western-blot. Condusion The HpI2 NP was expressed in E.coli successfully and can be used to produce polyclonal antibodies.
出处
《热带医学杂志》
CAS
2008年第4期340-343,共4页
Journal of Tropical Medicine
关键词
副流感病毒
核衣壳蛋白
基因克隆
基因表达
多克隆抗体
human parainfluenza virus
nucleocapsid protein
gene cloning
gene expression
muhiclonal antibodie
