诺瓦克病毒ORF2的原核表达及其产物分析

Expression of the ORF2 of Norovirus in Escherichia coli and the Product Analysis

摘要:

诺瓦克病毒(Norovirus, NV)是1972年在美国首次发现的新生病毒, 直到1995年国内才有报道。该病毒是严重危害人类健康的重要食源性病毒, 可导致不同年龄阶段人群的急性病毒性腹泻。实验以提取临床NV阳性腹泻样本的基因组RNA为模板, 采用RT-PCR方法扩增得到全长为1623 bp编码NV主要衣壳蛋白VP1的ORF2全基因序列。将测序结果进行进化分析, 结果表明该病毒属于NV GII。将ORF2亚克隆到原核表达载体pET-28a构建原核表达载体, 鉴定后转化大肠杆菌BL21(DE3)感受态细胞。经IPTG诱导表达, 获得大小约62 kD的融合蛋白并命名为rVP1。以纯化的rVP1免疫新西兰大白兔后制备高免血清, Western Blot结果表明, 实验所获得的多克隆抗体可以特异性识别临床样本中NV VP1。因此, rVP1具有良好的免疫原性和反应原性。对小于rVP1的蛋白条带进行Western Blot分析, 结果表明所有条带均为rVP1的部分蛋白。双向琼脂扩散试验结果显示实验所获得的高效价多克隆抗体与纯化的原核表达产物仅形成单一沉淀带。

Abstract:

Norovirus (NV) was one of new borne viruses, which was found firstly in the USA in 1972 and not reported in China until 1995. The main food-borne viral pathogens affect people badly and cause the epidemic acute gastroenteritis in all age groups worldwide. In this study, the genomic RNA was extracted from the non-bacterial gastroenteritis samples. A 1623 bp fragment, containing the complete coding sequence of the ORF2 gene, was amplified from the NV samples by RT-PCR. Sequencing analysis showed that it belongs to GII and shared more than 99% homology with the corresponding sequences published in the GenBank(DQ419908 and DQ369797). The ORF2 gene was then in-frame fused to the prokaryotic expression vector pET-28a, and the resultant recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells. A 62 kD fusion protein, named rVP1, was expressed after IPTG induction. Rabbits were immunized by the purified rVP1. Western Blot results showed that the high titer antibody can specifically recognize the VP1 in the clinical samples from hospital. The data suggested that the rVP1 has good immunogenicity and reactiongenicity. The minor protein in the expression product was analyzed by Western Blot and double?immunodiffusion?test. The data showed that the minor proteins were the fragments of rVP1.