鸭肠炎病毒UL6基因B细胞表位的预测及其主要抗原域的原核表达

采用DNAStar Protean程序,综合运用二级结构、亲水性、可塑性和抗原性指数等参数,对鸭肠炎病毒(DEV)CHv毒株UL6基因(GenBank登录号EF055890)的氨基酸序列进行了B细胞表位预测。结果显示,UL6蛋白羧基端第Thr507~Gln515、Thr521~Met529、Asp531~Phe539、Gly544~Asn562、Thr568~Gln585、Lys592~Val604、Ala681~Ala778和Tyr780~Lys790区域内含有优势B细胞表位。以DEVCHv毒株基因组作为PCR模板,利用Oligo6.0软件设计了1对引物,整体扩增涵盖上述具有优势表位的基因片段,将其亚克隆到原核表达载体pET-32a( )中,获得表达载体pET-32a-UL6M,再将其转化至E.coliRosetta(DE3),经IPTG诱导,外源基因获得了良好表达。以兔抗DEV多克隆血清进行Western-blot分析,抗血清可与该融合蛋白发生特异性反应。提示,该氨基酸片段可能含有软件预测的线性表位区。

Prediction of epitopes on B cell of UL6 gene of duck enteritis virus and prokaryotic expression of major antigen determinant sequence

Using DNAStar Protean software,the amino acid sequence encoded by UL6 gene(GenBank accession number EF055890) of duck enteritis virus(DEV) CHv strain was analyzed in secondary structure,hydrophilicity,flexibility and antigenic indexes to predict B cell epitopes.Many distinct antigen epitopes were situated in the C-terminal regions of Thr507-Gln515,Thr521-Met529,Asp531-Phe539,Gly544-Asn562,Thr568-Gln585,Lys592-Val604,Ala681-Ala778 and Tyr780-Lys790.A fragment encoding UL6 major epitopes was amplified by PCR and subcloned the sequence into the prokaryotic expression vector pET-32a( ).The recombinant plasmid pET-32a-UL6M was transformed into Escherichia coli Rosetta(DE3) and expressed under the induction of IPTG.Western-blot analysis indicated that polyclonal rabbit-anti-serum against DEV had specific reaction with the recombinant protein.These results indicated that the predicted antigenic region may contain the linear epitopes.