盖他病毒E2蛋白的表达、纯化以及RT-PCR检测方法的初步建立

发布时间：2018-01-26 03:45

本文关键词： 盖他病毒 E2基因原核表达 RT-PCR　出处：《湖南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：盖他病毒(Getah virus, GETV)属于披膜病毒科(Togaviridae)甲病毒属(Alphavirus)成员,它通过蚊虫叮咬脊椎动物的方式来传播疾病。GETV在猪群中流行会危害养猪产业,它被认为是引起母猪繁殖障碍的原因之一。GETV能引起母猪繁殖障碍,新生仔猪轻度发热、皮疹,后腿水肿且伴有精神沉郁和腹泻。到目前为止对GETV所引起的疾病仍然没有十分有效的治疗药物和疫苗。因此,建立特异性强、准确性高的检测方法就显得尤为重要。本实验参考GenBank (EU015066) Getah virus SH05-6中结构蛋白E2的全基因组序列,对其进行密码子优化后人工合成该基因。将合成的E2基因克隆至原核表达载体pCold I中,成功构建重组质粒pCold I-E2。通过对重组质粒pCold I-E2进行1PTG低温诱导蛋白表达,SDS-PAGE和Western Blot分析,结果可见在相对分子质量46KD处出现目的条带,与预期相符,且蛋白表达量占大肠杆菌表达总量的80%,E2基因在大肠杆菌低温表达系统中获得了高效表达。对E2蛋白进行纯化,并优化了纯化条件,最终获得了高纯度的盖他病毒E2蛋白,并且制备了抗E2蛋白多克隆抗体。以E2基因为模板,设计特异性引物,优化反应条件,初步建立了用于检测GETV的RT-PCR方法,使用该方法检测蚊虫体内所携带的GETV。检测结果显示从上海地区所采集的蚊虫体内并未检测到GETV。本实验对我国GETV快速诊断技术的研究和发展具有重要推动作用。
[Abstract]:Getah virus (GETV) is a member of the family TogaviridaeA belonging to Alphavirus. It spreads the disease through mosquitoes biting vertebrates. GETV is prevalent among pigs and can harm the pig industry. It is considered to be one of the causes of reproductive disorders in sows. GETV can cause reproductive disorders in sows, mild fever and rash in newborn piglets. Hind leg edema accompanied by depression and diarrhea. So far the disease caused by GETV is still not very effective treatment drugs and vaccines. Therefore, the establishment of a strong specificity. The accuracy of the detection method is particularly important. This experiment refers to GenBank / EU015066). The whole genome sequence of structural protein E2 in Getah virus SH05-6. The synthesized E2 gene was cloned into prokaryotic expression vector pCold I after codon optimization. The recombinant plasmid pCold I-E2 was successfully constructed. The recombinant plasmid pCold I-E2 was expressed by 1PTG hypothermia induction protein. The results of SDS-PAGE and Western Blot analysis showed that the target band appeared at 46KD relative molecular weight, which was consistent with the expectation. The E2 gene of 80% of the total expression amount of E. coli was highly expressed in the low temperature expression system of Escherichia coli. The E2 protein was purified and the purification conditions were optimized. Finally, a high purity E2 protein of capsid virus was obtained, and the polyclonal antibody against E2 protein was prepared. Using E2 as template, specific primers were designed to optimize the reaction conditions. A RT-PCR method for detecting GETV was preliminarily established. This method was used to detect the GETV carried in mosquitoes. The results showed that no GETV was detected in mosquitoes collected from Shanghai area. The study on the rapid diagnosis of GETV in China was conducted in this experiment. The exhibition plays an important role in promoting it.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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