马疱疹病毒1型ORF30基因原核表达载体的构建

马疱疹病毒1型(Equid herpesvirus 1,EHV-1)是引起孕马流产的主要病原体之一,也与死胎、新生幼驹死亡、青年马鼻肺炎以及一种名为马疱疹病毒性脑脊髓病(Equine herpesvirus myeloencephalopathy,EHM)的神经系统疾病有关。近些年来,EHM出现的频率越来越高,对养马业和赛马业的危害很大。最新研究结果显示,神经致病性毒株与编码病毒DNA聚合酶催化亚基的ORF30基因单核苷酸点突变有着很高的相关性。为了获得马疱疹病毒1型DNA聚合酶,并制备DNA聚合酶的多克隆抗体,本研究以EHV-1-XJ2015毒株基因组DNA为模板,扩增ORF30基因编码区,并连接于pMD19-T载体上,用BamHI和HindIII双酶切后,将酶切产物插入表达pET-28a(+)中,转化入大肠杆菌DH5感受态细胞,构建ORF30基因的原核表达载体pET-28a(+)-ORF30。通过酶切、PCR扩增和测序等方法对阳性重组质粒进行了鉴定。结果表明,构建的重组质粒pET-28a(+)-ORF30经BamhⅠ和HindⅢ双酶切后,经1%琼脂糖凝胶电泳,电泳结果与预期相符,表明目的片段成功插入载体pET28a(+)中。通过采用特异性引物对重组质粒pET28a(+)-ORF30进行PCR鉴定,产物经1%琼脂糖凝胶电泳观察,得到一条大小约1074bp的清晰条带,与目的基因大小相符,表明重组质粒pET28a(+)-ORF30构建成功。本研究为进一步探讨马疱疹病毒1型ORF30基因遗传变异对DNA聚合酶功能的影响奠定了基础。

Construction of Prokaryotic Expression System of ORF30 Gene of Equine Herpesvirus Type

Equine herpesvirus type 1 (EHV-1) is one of the main infectious causative agents of abortion in mares, which is associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). In recent years, EHM is increasing,resulting in harm to horse breeding and horse racing. As it was founded that the neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carried a single nucleotide point mutation in the ORF30, which encoded a catalytic subunit of viral DNA polymerase. In order to obtain DNA polymerase of equine herpes virus type 1 and to make polyclonal antibodies to DNA polymerase, the genomic DNA of EHV-1-XJ2015 virus strain was used as the template, then the ORF30 gene coding region was amplified and connected to pMD19-T vector. After bienzyme digestion with BamH I and Hind III, the enzyme digestion products were inserted into the expression of pET-28a(+) and transformed into e. coli DH5 competent cells. The prokaryotic expression vector pET-28a(+)-ORF30 was constructed. The positive recombinant plasmid was identified by enzyme digestion, PCR amplification and sequencing. Results showed that the constructed recombinant plasmid pET-28a(+)-ORF30 via BamhⅠand Hind Ⅲ double enzyme, after a 1% agarose gel electrophoresis, the electrophoresis results with expectations, that purpose fragment successfully inserted into the carrier pET28a (+). The recombinant plasmid pET28a(+)-ORF30 was identified by PCR using specific primers. The product was observed by 1% agarose gel electrophoresis, and a clear band of about 1074 bp was obtained, which was consistent with the size of the target gene, indicating that the recombinant plasmid pET28a(+)-ORF30 was successfully constructed. In this study, a foundation was laid for further study on the effect of genetic variation of equine herpes virus type 1 ORF30 gene on the function of DNA polymerase.