摘要
为进一步建立Ⅰ群血清4型禽腺病毒（FADV-4）的特异性检测方法,根据本实验室分离鉴定的安卡拉病毒的基因组序列,设计针对六邻体蛋白（Hexon）基因的特异性引物,经PCR扩增,连接至p ET-28a中,构建原核表达载体p ET-28a-Hexon。将其转化感受态BL21中,经异丙基硫代半乳糖苷（IPTG）诱导,SDS-PAGE分析,获得重组蛋白大小为49 ku。Western blot分析纯化后重组蛋白能与FAd V-4阳性血清发生特异性反应。将纯化后的重组蛋白免BALB/c小鼠,制备了六邻体蛋白多克隆抗体,采用间接ELISA方法测定效价表明制备的血清效价大于1∶64 000。
Based on the group Ⅰ fowl adenovirus serotype 4（FADV-4） genome sequence,a pair of specific primers were designed to amplify the hexon gene by PCR,and then cloned into the p ET-28 a vector named p ET-28 a-Hexon. The recombinant plasmid was transformed into competent cells BL21 induced with IPTG for expression. The results showed that 49 ku of the fusion proteins was obtained following an SDS-PAGE analysis. Expressions induced with different IPTG concentrations,temperatures and durations suggested the best IPTG induction concentration to be 1 mmol/L,the optimum temperature to be 37℃,and the optimal duration of time to be 4 h. Western-bolt analysis showed that the recombinant fusion proteins reacted specifically with the FAd V-4 positive serum. The purified recombinant proteins were inoculated in BALB/c mice to prepare polyclonal antibody against hexon protein. The titer of the polyclonal antibody was found to be higher than 1 ∶ 64 000,assayed by indirect ELISA. The high efficiency single factor serum prepared in this experiment laid the foundation for the study on the specific detection of serotype 4 adenovirus.
出处
《畜牧与兽医》
北大核心
2018年第2期83-85,共3页
Animal Husbandry & Veterinary Medicine
关键词
Ⅰ群4型禽腺病毒
六邻体蛋白
多克隆抗体
group Ⅰ fowl adenovirus serotype 4
hexon protein
polyclonal antibody
