摘要
为对微小牛蜱烯醇酶(Enolase)基因进行真核表达及免疫反应原性分析,本研究采用RT-PCR方法扩增微小牛蜱Enolase基因的开放阅读框(ORF)序列,将其克隆至pFastBac1载体中构建重组质粒pFastBac1-Enolase,随后转化至大肠杆菌DH10Bac细胞中制备了重组杆粒Bacmid-Enolase,转染至SF9细胞以获得重组Enolase蛋白。通过双酶切、PCR及测序鉴定显示Enolase基因正确插入。利用SDS-PAGE分析表达产物,结果显示目的蛋白大小约为48 ku。利用western blot对重组蛋白进行分析,结果表明目的蛋白能被微小牛蜱抗原免疫兔阳性血清识别,具有良好的免疫反应原性。凝血酶时间(TT)的测定结果显示,Enolase重组蛋白能显著延长TT,具有抗凝血活性。本研究首次采用Bac-to-Bac杆状病毒表达系统对微小牛蜱Enolase基因进行真核表达,并鉴定了重组蛋白的免疫反应原性,为开展Enolase免疫原性分析及功能研究奠定了基础。
In order to perform eukartoyic expression and immunogenicity analysis of the Enolase gene of Rhipicephalus microplus, open reading frame(ORF) of Enolase gene of R. microplus was amplified by RT-PCR. The Enolase gene was cloned into pFastBac1 vector to construct the recombinant plasmid(pFastBac1-Enolase). After transforming E. coli DH10 Bac competent cells, the recombinant Bacmid-Enolase was prepared and transfected into SF9 cells to obtain recombinant protein. Double digestion, PCR and sequencing analysis showed that the Enolase gene was inserted into baculovirus correctly. SDS-PAGE analysis showed that the recombinant protein Enolase was detected with the size of 48 ku. Western-blot assay was used to analyze the recombinant protein, and the results showed that the target protein can be recognized by the rabbit anti-R. microplus serum and possessed good immunoreactivity. The results of thrombin time(TT) showed that Enolase recombinant protein could significantly prolong TT and the recombinant protein had anticoagulant activity. In this study, eukaryotic expression of the R. microplus Enolase gene was carried out using Bac-to-Bac baculovirus expression system and the immunoreactivity of the recombinant protein was identified for the first time, laying a foundation for the development of Enolase immunogenicity analysis and functional research.
作者
徐兴莉
杨虎
XU Xing-li;YANG Hu(College of Life Science and Resource Environment,Yichun University,Yichun 336000,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第8期890-894,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
江西省科技厅自然科学基金项目(20181BAB214017)
江西省教育厅自然科学基金项目(GJJ161011)
国家自然科学基金(31960697)。
