禽腺病毒4型鞭毛重组蛋白的原核表达及其免疫效果研究

Study on expression of recombinant flagellin-fiber2 protein and its immunoprotective effects

为研究禽腺病毒4型重组鞭毛-fiber2蛋白免疫保护性,将鼠伤寒沙门菌鞭毛蛋白基因与禽腺病毒4型的fiber2基因融合,再将融合基因和单独的fiber2基因分别克隆到原核表达载体p ET-28a,后将构建的p ET-28a-flagellin-fiber2和p ET-28a-fiber2重组载体分别转入大肠杆菌BL21细胞,经IPTG诱导表达,利用SDS-PAGE和Western Blot鉴定表达产物,再将表达产物纯化后免疫21日龄SPF鸡;结果表明,重组菌在37℃培养条件下1.0 mmol/L的IPTG诱导6 h可表达最多的可溶性重组蛋白,Western Blot分析表明,所表达的重组蛋白均能与禽腺病毒血清4型感染阳性血清发生特异性反应,说明所表达蛋白具有良好反应原性,动物试验表明,表达的两种目的蛋白能为鸡提供一定保护力,且低剂量的重组鞭毛-fiber2蛋白产生较好的免疫保护效果。

Tbe study was to investigate the immunoprotective effects in specific pathogen free （SPF） chickens for recombinant flagnllin-fiber2-protein of fowl adenovirus （FAdV） serotype 4. A recombinant gene that was designed by fusing a modified version of Salmonella typhimurium flagellin to FAdV-4 fiber2 gene and a fiber2 gene were cloned into the pET-28a vector for expression in Esch- edchia coli BL21. The recombinant proteins were analyzed by SDS-PAGE and Western Blot,and then were used to immunize 21 day-old SPF chickens. The recombinant proteins were induced with 1 mmo]/L isopropyl 15-D-thiogalactoside （IPTG） for 6 h at 37℃. Western Blot results showed that the proteins had specific reaction against FADV-4 positive serum,implying that these proteins had good reactivity with the positive serum. Animal test results indicated that the two proteins protect SPF birds from challenge. Moreo- ver, the protective effect of low-dose recombinant flagellin-fiber2 protein was better.