DNA vaccine constructs against enterovirus 71 elicit immune response in mice.
DNA vaccine constructs against enterovirus 71 elicit immune response in mice.
复制标题
DOI：
10.1186/1479-0556-5-6
复制
发表时间：
2007-04-19
期刊：
影响因子：
--
通讯作者：
Rosli R
中科院分区：
文献类型：
--
作者：
Tung WS;Bakar SA;Sekawi Z;Rosli R
肠病毒71型（EV71）是导致儿童和婴儿常见皮疹疾病——手足口病（HFMD）大规模暴发的主要病原体。对于严重的EV71感染没有有效的抗病毒治疗，也没有疫苗。本研究的目的是利用EV71病毒衣壳蛋白（VP1）基因设计和构建一种抗EV71病毒的DNA疫苗，并验证该DNA疫苗在体外和体内的功能。用PCR扩增了两株本地爆发株EV71病毒的VP1基因，并将其插入真核表达载体pVAX1中。利用PCR、酶切分析和DNA测序等方法筛选阳性克隆。然后测试构建物在Vero细胞中的蛋白表达。随后，在体内研究中，用DNA疫苗构建物免疫雌性Balb/c小鼠。采用酶联免疫吸附法（ELISA）和病毒中和法检测小鼠体内抗vp1 IgG的存在及其对EV71病毒的中和作用。pVAX1载体克隆成功，每个分离物（S2/86/1和410/4）的VP1基因在正确的方向和帧内克隆成功。含有VP1基因的DNA疫苗构建体在体外无细胞表达系统中得以表达。VP1蛋白在哺乳动物细胞系中成功表达，并通过RT-PCR、间接免疫荧光法（IFA）和western blotting对VP1蛋白进行检测。用DNA疫苗结构免疫小鼠的抗vp1 IgG水平在第一次增强后升高，但在第二次增强后下降。用DNA疫苗结构免疫小鼠的抗vp1 IgG对EV71表现出中和活性。本研究获得的有希望的结果促使进一步的测试，以提高这种潜在的EV71 DNA疫苗的表达和免疫原性。
Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo. The VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71. The pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71. The promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine.