应用酵母双杂交系统初步筛选IBDV VP2结合蛋白

Preliminary Screening Candidate IBDV VP2-binding Proteins by Yeast Two-hybrid System

用RT-PCR扩增VP2 cDNA并克隆入诱饵载体pSos,与鸡法氏囊cDNA文库中分离的质粒DNA共转化cdc25H,筛选阳性酵母克隆;用酵母质粒DNA转化大肠杆菌,提取质粒再分别与诱饵载体pSosVP2共转化酵母细胞,利用酵母双杂交系统对阳性克隆进行验证。对阳性克隆的插入片段进行序列分析,根据生物信息学分析结果初步确定IBDV VP2结合蛋白编码基因。结果显示,经酵母双杂交共筛选出48个候选阳性克隆,分别转化酵母菌,经酵母双杂交验证后,获得阳性克隆19个。进一步的序列分析显示,在19个插入序列中,共编码8种不同的多肽,分别是Ras超家族中的RRAS2、Rit1和N-Ras,肿瘤相关蛋白TCTP和PARP14,抗病毒Mx蛋白,LAMB3蛋白以及蛋白激酶PRKX。VP2候选结合蛋白的获得为IBDV受体、诱导细胞凋亡或逃逸宿主抗病毒机制等研究奠定了基础。

Our study was conducted to isolate the candidate binding proteins of IBDV VP2. The VP2 cDNA was amplified by RT-PCR and subcloned into the bait vector pSos in yeast two-hybrid system. The plasmid DNAs from chicken bursal cDNA library were inserted into the prey vector. The recombinant bait and prey vectors were co-transformed into cdc25H for cDNA library screen- ing using the yeast two-hybrid system. After first round of screening, 48 putatively positive clones were obtained, among of which 19 clones were confirmed to be positive by the second round of screening. Among the 19 positive clones, 8 encoded polypeptides shared high homolo- gies with known proteins, the Ras subfamily such as RRAS2, Ritl and N-Ras, the TCTP and PARP14 are tumor proteins, Mx have antivirus activity, PRKX is a protein kinase, and LAMB3. The finding of candidated VP2-binding proteins made foundation for research on IBDV receptor （s）, apoptosis or the pathgenic merchanism of IBDV.