摘要
目的构建pET-22b-ECVP1表达载体,在大肠杆菌中诱导表达肠道病毒71型(EV71)及柯萨奇B3型(CB3)病毒VP1融合蛋白(ECVP1),并鉴定其抗原性。方法利用逆转录聚合酶链式反应(RT-PCR)方法分别从EV71与CB3病毒基因组中扩增得到外壳蛋白VP1基因,经重叠延伸PCR(SOE-PCR)方法拼接构建重组融合蛋白基因(ecvp1),将该片段连接到pET-22b表达载体上,在大肠杆菌BL21(DE3)中诱导表达融合蛋白,经Western blot鉴定其抗原性。结果pET-22b-ECVP1表达载体构建成功,融合蛋白相对分子量约为65 ku,Western blot分析表明,该融合蛋白能被抗EV71及抗CB3抗体特异识别。结论成功构建pET-22b-ECVP1表达质粒并诱导ECVP1融合蛋白表达,且融合蛋白具有良好的抗原性。
Objective To construct the pET-22b-ECVP1 recombinant plasmid and express the fusion protein VP1 s of EV71 and CB3 in E. coli and identify its antigenecity. Methods The sequences encoding VP1 were separately acquired by RT-PCR from the genome of EV71 and CB3. Then fusion sequence of ECVP1 was obtained by SOE-PCR. The sequence was cloned into pET22b, and the recombinant plasmid was transformed into E. coli BI21 （DE3） strains. Then strains were incubated for expression, the fusion protein was identified by Western blot. Resuits The expression plasmid of pET-22b-ECVP1 was constructed successfully, the molecular weight of ECVP1 protein were about 65 ku and later fusion protein ECVP1 was identified with anti-EVTl and anti-CB3 by Western blot. Conclusion Fusion protein vector of pET-22b-ECVP1 has been obtained, ECVP1 fusion protein expressed successfully, and the fusion protein has antigenicity.
出处
《安徽医科大学学报》
CAS
北大核心
2013年第6期583-586,共4页
Acta Universitatis Medicinalis Anhui
基金
北京市自然科学基金(编号:7112107)
