共表达口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因牛肾细胞株的筛选及稳定性

Screening and stability of Madin-Darby bovine kidney cell strainco-expressing the capsid precursor protein P1-2A gene and the protease 3C gene of Foot-and-mouth Disease Virus

摘要:

摘要：【目的】筛选稳定表达口蹄疫病毒衣壳蛋白的牛肾细胞（Madin-Darby bovine kidney，MDBK）株。【方法】采用聚合酶链式反应（Polymerase chain reaction，PCR）方法从重组质粒pMD-P1-2A和pMD-3C中分别扩增口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因，将两基因依次插入逆转录病毒载体pBABE-puro。重组逆转录病毒载体pBABE-puro/P1-2A-3C和pVSV-G质粒载体用脂质体介导共转染GP2-293包装细胞。产生的重组逆转录病毒感染MDBK细胞后使用嘌呤霉素筛选抗性细胞。利用克隆环套取法得到单克隆细胞。经间接免疫荧光和酶联免疫吸附测定（Enzyme-linked immunosorbent assay，ELISA）方法检测MDBK细胞中衣壳蛋白的表达，并在电镜下观察口蹄疫病毒空衣壳。【结果】成功筛选到稳定表达口蹄疫病毒衣壳蛋白的MDBK细胞株，衣壳前体蛋白P1-2A在蛋白酶3C裂解作用下正确组装成空衣壳。【结论】该研究为口蹄疫亚单位疫苗的研制提供了实验材料。

Abstract:

Abstract: [Objective] To screen the Madin-Darby bovine kidney cell strains for stable expression of capsid protein of foot-and-mouth disease virus (FMDV ). [Methods] We obtained two genes coding for capsid precursor protein (P1-2A) and protease (3C) of FMDV by PCR from recombinant plasmids pMD-P1-2A and pMD-3C.The recombinant retroviral vector pBABE-puro/P1-2A-3C was constructed by inserting P1-2A gene and 3C gene into pBABE-puro. Both the recombinant plasmid pBABE-puro/P1-2A-3C and the plasmid pVSV-G were co-transfected into packaging cells GP2-293 by liposome-mediated transfection method. As a result, the recombinant pseudovirus was produced. The pseu-dovirus infected the interesting target cell MDBK. The infected MDBK cells were selected by puromycin(2.5 mg/mL) for 2 weeks. The monoclonal cells were selected using cloning rings. The expression of capsid protein was detected by indirect immunofluorescence and sandwich-ELISA. The empty capsids of FMDV were observed under electron microscope. [Re-sults] The capsid protein of FMDV was expressed in MDBK cells. The expression levels in screened cell strains of various passages showed no significant difference. [Conclusion] The MDBK cell strains for stable expression capsid protein of FMDV were successfully screened, which laid a foundation of development of FMDV subunit vaccine.