禽脑脊髓炎病毒VP3基因的克隆与表达

Cloning and Sequencing of VP3 Structural Protein Gene of Avian Encephalomyelitis Virus

目的对VP3进行克隆和测序后,在杆状病毒系统中表达获得重组蛋白,为AEV的分子诊断以及更深一层次的分子和基因工程研究打下基础。方法利用RT_PCR技术,从AEV VR株感染的SPF鸡胚脑及内脏器官组织中提取病毒RNA并扩增出VP3目的基因,而后克隆到pMD18-T载体中,鉴定后进行序列测定;应用Bac_to_Bac杆状病毒表达系统,将VP3基因定向克隆到pFastBacHTa donor质粒中,经转座反应,筛选到VP3_Bacmid重组子,转染sf9昆虫细胞并盲传三代,提取DNA应用PCR方法鉴定证实获得含VP3的重组病毒,Western blot鉴定VP3在杆状病毒系统中得以表达。结果AEV VR株VP3基因全长735 bp,共编码245个氨基酸,与AEV_1143标准毒株相应片段的核苷酸和氨基酸同源性分别为93%和97%;并将其与其它小RNA病毒进行了比较;Western blot鉴定结果,重组蛋白大小约27 ku。结论本研究通过RT_PCR技术首次从体外成功扩增AEV Van_Roekel株VP3蛋白基因,并对其进行克隆、测序;应用Bac_to_Bac杆状病毒表达系统成功地表达了VP3基因并获得了具有良好免疫活性的重组蛋白。

Objective In order to make a foundation for molecular diagnosis and gene engineering studies of Avian encephalomyelitis virus （AEV）, the structural protein VP3 gene of AEV and expression of its recombinant protein were cloned and sequenced. Methods We designed a pair of primers to amplify the van-Roekel VP3 gene from AEV infected SPF embryo with specific RT-PCR technique. The amplified product was cloned into PDM18-T vector after purification and withdraw. The VP3 gene was sub-cloned into the eucaryotic expression donor plasmid pFastHTa, which was also digested by EcoR I and Xho I. The recombinant plasmid was transfected into E. coll. DH10Bac competence cells. Then selected by PCR and identified by EcoR I and Xho I degistion, and transposition happened. The transpositional recombinant bacmid was transfected into Sf9 insect cells, and then a kind of recombinant baculovirus was obtained. The antigen activity of the recombinant protein was identified by Western blot. Results The results showed that VP3c contained 735 bp and 245 amino acids, sharing a 93 % and 97 % homology with AEV-1143 strain, respectively. It was also compared with other small RNA viruses, and recombinant protein was also achieved by Western-blot. The recombinant protein was about 27 ku. Conclusion Using RT-PCR technique, the van-Roekel VP3 gene has been for first time amplified in vitro. It has been also cloned and sequenced. Using Bac-to-Bac expression system, the recombinant protein has been achieved and proven to exhibit high immune activity.