鸭肠炎病毒gL蛋白在鸡胚成纤维细胞中动态表达的检测

为研究鸭肠炎病毒(DEV)gL蛋白在感染鸡胚成纤维细胞(CEF)过程中的表达情况,本研究以DEVClone-03基因组为模板,应用PCR方法分别扩增得到截短的(gLt,181 bp～711 bp)和全长的gL(1 bp～711 bp)两个基因片段。将gLt基因片段克隆至pET-30a原核表达载体,转化E.coli BL21(DE3),经IPTG诱导表达并对表达产物进行纯化复性,免疫BALB/c小鼠,制备鼠抗gL蛋白多克隆抗体。同时将全长gL基因克隆至真核表达载体pcDNA3.1(+),构建真核表达重组质粒pcDNA-gL,转染293T细胞。采用获得的抗gL蛋白抗体检测DEV感染CEF后及真核表达质粒pcDNA-gL转染293T细胞后gL蛋白在不同时间点的表达情况。结果表明,在pcDNA-gL转染293T细胞后12 h应用western blot方法能够检测到gL蛋白的表达,其表达量随着转染时间增加而增加;在病毒感染CEF后24 h应用间接免疫荧光方法能够检测到gL蛋白少量的表达,western blot方法在病毒感染CEF48 h后检测到gL蛋白的表达,其表达量随着病毒感染时间增加而增加。上述结果提示,编码gL蛋白基因可能是病毒复制的晚期表达基因。

Detection of the duck enteritis virus gL protein expressed in chicken embryo fibroblast

To study the expression of gL protein in chichen embryo fibroblast(CEF) infected by duck enteritis virus(DEV),the truncated gL(gLt,181 bp-711 bp) and whole gL(1 bp-711 bp) gene were amplified by PCR from DEV genome,respectively.The gLt fragment was cloned into pET-30a and transformed into E.coli BL21(DE3).The recombinant gLt protein was expressed with IPTG induction and purified by ProBondTM Purification System.The specific antiserum against gL was produced in mouse immunized by the gLt protein.While,the gL gene was cloned into the pcDNA3.1(+) and transfected into 293T cells.The results indicated that expression of gL protein was detected in 293T cells as early as 12 hours post transfection by western blot and increased thereafter.However,the gL protein was detected in DEV infected CEF at 24 hours by indirect immunofluorescent assay and at 48 hours post infection by western blot,and increased in a time-dependent manner.The results suggested that the encoding gene of gL was a late gene during DEV replication.