鸭肠炎病毒UL53截段基因的序列特性、原核表达与其抗原性检测

Sequence Characteristics,Prokaryotic Expression as Well as Antigenicity Detection of the Truncated UL53 Gene of Duck Enteritis Virus

【目的】通过选取DEV-UL53基因主要抗原域与进行DEV-UL53截段基因B细胞表位多参数预测相结合的策略,高效表达DEV-UL53截段基因,并通过Westernblot分析重组蛋白的免疫原性。【方法】通过生物信息学软件DNAStarProtean模块对UL53基因编码的gK蛋白进行主要抗原域预测,选取主要抗原域对应的UL53截段基因进行二级结构、蛋白质骨架柔性区域、表面可及性区域预测和在线预测该蛋白的亲水性及跨膜区,并对UL53截段基因进行克隆、亚克隆、原核表达与抗原性分析。【结果】UL53截段基因编码蛋白gK的B细胞表位最可能分布于Ala20—Leu25、Ser40—Met47、Leu68—Ile78、Val124—Phe128、Ile129—Tyr134、Asp176—Ile178,构建的阳性表达质粒转入BL21表达宿主菌经IPTG诱导外源基因获得了良好表达,经Westernblot分析表明该重组蛋白具有良好的抗原性。【结论】实现了UL53截段基因的高效表达,经Westernblot分析表明该重组蛋白具备良好的免疫原性,这为DEV-UL53基因及其编码的蛋白gK功能的深入研究、新型疫苗和诊断试剂的研制及开发等提供可用的试验材料。

【Objective】 Applying the strategy to select the main antigenic domains of DEV-UL53 gene and carry out multiparameter B cell epitope prediction of DEV-truncated UL53 gene, to express efficiently the DEV-truncated UL53 gene and analyze the antigenicity of this fussion protein by Western blot. 【Method】 The bioinformatics software DNAStar Protean was used to predict the main antigenic domains of gK protein coded by DEV-UL53 gene, then the truncated UL53 gene corresponding the main antigenic domains was selected and used to predict the secondary structure, flexibility domains, surface possibility with DNAStar software and predict hydrophilicity as well as transmembrane domains on-line, meanwhile clone, subclone as well as prokaryotic expression of the truncated UL53 gene, also the antigenicity of the fussion protein was detected by Western blot. 【Result】 The B cell epitopes of gK coded by DEV-truncated UL53 gene most likely distribute in Ala20—Leu25, Ser40—Met47, Leu68—Ile78, Val124—Phe128, Ile129—Tyr134, Asp176—Ile178. The recombinant expression plasmid was transformed into BL21 and expressed under the induction of IPTG. Western blot analysis indicated that polyclonal rabbit antiserum against DEV had specific reaction with the fussion protein. 【Conclusion】 DEV-truncated UL53 gene was efficiently expressed in prokaryotic system and also the gK fussion protein has good reactionogenicity, which provided useful data for research of the UL53 gene, gK protein and development of new generational vaccine and diagnostic reagent.