J亚群禽白血病毒gp85基因克隆表达与初步应用

在本实验室分离鉴定J亚群禽白血病病毒的基础上,用PCR方法扩增gp85基因,长度为921 bp的DNA片段,与J亚群禽白血病标准毒株序列比对同源率为96.4%。将该片段连接到pET28a表达载体上,构建了重组表达质粒pET28a-S1-Jgp85,对质粒测序后分析后,转化大肠杆菌BL21(DE3),用IPTG诱导表达,经SDS-PAGE分析,表达出约为40 kD的融合蛋白。Western blot结果表明,表达产物可以与ALV-J阳性血清发生特异性反应。以纯化的His-Jgp85重组蛋白为包被抗原,建立间接ELISA方法,用于J亚群禽白血病的临床诊断。本研究对抗原包被浓度,待检血清稀释浓度,酶标抗体浓度,特异性试验及临界值等条件进行优化,对优化后的ELISA检测方法进行了临床应用试验。本研究建立了能特异性检测J亚群禽白血病血清抗体的ELISA方法。

Cloning,expression and application of gp85 gene from subgroup J avian leukosis virus(ALV)

Basing on isolation and identification of subgroup J avian leukosis virus,a DNA fragment of 921 bp encoding gp85 of subgroup J avian leukosis virus was amplified by PCR.The gene was then cloned into pET28a expression plasmid.After sequencing and alignment with the standard strain,the recombined plasmid was transformed into E.coli BL21(DE3) and induced by IPTG.SDS-PAGE analysis showed that the fusion protein of gp85 was efficiently expressed and the protein size was about 40 KD.The result from Western blot s...