镰形扇头蜱组胺结合蛋白RhHBP基因的克隆和重组表达

为了进行抗蜱及蜱传病疫苗的研究,本试验根据GenBank中的镰形扇头蜱组胺结合蛋白(Rhipicephalus haemaphysaloideshistamine bindingprotein,RhHBP)基因cDNA序列,设计一对特异性引物(内含EcoRI/XhoI酶切位点),经RT-PCR扩增了镰形扇头蜱组胺结合蛋白RhHBP的cD-NA,大小为603 bp,将其克隆到pGEM-Teasy中并测序。在去除编码RhHBP信号肽的核苷酸序列后,将其亚克隆到pGEX-4T-1中,构建重组质粒pGEX-4T-1-RhHBP,将该重组质粒转化大肠杆菌BL21,经终浓度为1 mmol/L IPTG诱导其表达,表达的融合蛋白大小为49 ku,以包涵体的形式存在。Western Blot分析表明,该蛋白具有良好的免疫原性。

Cloning and expression of histamine binding protein gene of Rhipicephalus haemaphysaloides

According to the Rhipicephalus haemaphysaloides histamine binding protein(RhHBP) gene sequence available in GenBank,the specific primers with EcoRI/XhoI site were designed by DNAMAN software.The RhHBP gene fragment was amplified by RT-PCR from genomic RNA and cloned into pGEM-T easy plasmid vector for sequencing.The RhHBP gene is 603bp in length.The gene fragment without the signal peptide was subcloned into vector pGEX-4T-1,and expressed in E.coli BL21 with the induction of 1mmol/L IPTG.The expressed protein had a molecular weight of 49 ku.The Western blot analysis indicated that the protein could react with the serum to RhHBP.