禽脑脊髓炎病毒VP1基因的克隆及表达

Cloning and expression of VP1 gene of avian encephaleomylitis virus

应用RT-PCR方法,扩增出AEV Van-roekel株结构蛋白基因VP1,测序结果表明VP1基因全长810 bp,编码270个氨基酸;和AEV 1143株相比,核苷酸同源性为94.2%,氨基酸同源性为99.26%。将VP1基因定向克隆到pGEX-6p-1载体谷胱苷肽转移酶基因的下游,并把重组质粒转入宿主菌BL21中,经IPTG诱导后,VP1基因得以表达。表达产物经SDS-PAGE和Western Blot鉴定,确定表达的融合蛋白大小约为58 Ku,且具有良好的反应原性。将表达产物纯化后免疫试验兔,琼脂扩散试验显示免疫血清能与禽脑脊髓炎琼脂扩散标准阳性抗原呈特异反应,表明重组VP1蛋白保留了天然蛋白的部分活性。

The structural protein VP1 gene of AEV strain was amplified and sequenced. The result of nucleotide sequencing showed VP1 gene consists of 810 nucleotides and it has the potential to encode 270 amino acids and to be 94.2 % nucleotides and 99.26 % amino acids identity with strain 1 143. To express VP1 in E. coli , a recombinant expression vector was constructed and transforrned into E. coli , then induced by IFIG at 37℃. SDS-PAGE and Western-blotting analysis showed the recombinant fusing protein had a molecular weight of approximately 58 Ku and reacted on antisera against AEV. The fusion protein was injected into rabbit to identify its antigenicity and the result was positive in AGP assary.