禽脑脊髓炎病毒Van Roekel株衣壳蛋白基因原核表达及其ELISA检测研究

利用RT PCR方法扩增了禽脑脊髓炎病毒VP0、VP3和VP1基因 ,分别插入pGEX 4T 1原核表达载体 ,转化到E .coliER2 566表达菌内 ,经IPTG诱导表达后 ,SDS PAGE分析表达产物。结果表明 ,表达的VP0、VP3、VP1融合蛋白分子量分别为 53 ,53 ,56ku。以电洗脱法纯化VP0、VP3、VP1表达蛋白抗原并分别建立了间接ELISA方法 ,用阳性血清和阴性血清进行检测。结果显示 ,表达VP1蛋白反应活性相对高于VP0、VP3蛋白反应活性 ,最后再将自制VP1板用自配试剂检测其活性 ,同时用美国试剂盒检测VP1活性 ,美国ELISA试剂盒、琼脂扩散试验检测同样 1 0份血清作比较 ,三组ELISA检测结果 ,其中有 8份被检血清为阳性 ;2份被检血清为阴性。这一结果与琼脂扩散试验检测结果一致。说明VP1表达蛋白活性达到预期要求 ,可以用做AE的ELISA诊断

The Expression of VP0,VP3,VP1 Gene of Avian encephalomyelitis virus Van Roekel Strain and Application in ELISA

The VP0,VP3,VP1 genes of Avian encephalomyelitis virus Van Roekel strain were successfully cloned and VP0,VP3,VP1 expressing plasmids were consturcted by inserting the target genes into pGEX-4T-1 vectors respectively.The VP1 expression proteins were analysed by ELISA. The result showed that expressed VP1 protein reacted with AE positive serum more stronger than the other two. An indirect ELISA method was set up using purified VP1 protein. as antigen and tested 10 serum samples. The result shown that 8 samples of them were positive,their S/P level are higher than 0.2.At the same time,10 serum samples were detected by ELISA kit and AGP.The results of detection showed that 8 of them were positive,but others are negative.This proved that the activity of expression protein satisfied the anticipative need.The result showed, this method can be used for diagnosis of AE.