摘要
从口蹄疫病毒(foot-and-m ou th d isease v irus,FM DV)细胞培养物中提取总RNA,经一步法RT-PCR获得了长约230 bp的3B基因片段,将PCR产物连接到pM D-18T载体上并测序,用B amHⅠ与H indⅢ双酶切后,将3B基因融合到pGEX-KG载体上形成表达质粒pGEX-KG-3B,转化到BL 21(DE 3)中在27℃诱导表达,将表达产物进行SDS-PAGE和蛋白质斑点EL ISA。结果表明3B基因主要以可溶形式高效表达,表达产物具有免疫原性。以纯化的表达产物为抗原建立了间接酶联免疫吸附试验(I-EL ISA)。方阵滴定其最佳包被浓度是6.1μg/孔,最佳血清稀释倍数为1∶80,通过测定36份FM DV阴性血清,确定了该方法的阳性判定标准。结果表明,该方法特异性强、重复性好;用3B-EL ISA方法与美国联合生物医学公司(U B I)生产的合成肽检测试剂盒U B I○RFM DV N S-EL ISA对比检测44份血清样品,符合率为93.1%。证明3B-EL ISA方法可用于口蹄疫的鉴别诊断。
The cDNA of 3B gene was synthesized using template RNA extracted from FMDV infected cells. The 3B gene was amplified by RT-PCR and subcloned into the expression vector,named as pGEX-KG-3B,followed by transformation of E. coli and identification. Under low temperature,the 3B protein expression was induced with IPTG,analyzed by SDS-PAGE and Protein Dot Blotting. The results showed that the 3B gene was successfully expressed in E. coli and the product possessed biological activity. The 3B-ELISA was developed and optimized using the purified fusion protein. The cut-off value was determined by testing 36 FMDV antibody negative sera. The 3B-ELISA is of good stability and specificity. Comparison between 3B-ELISA with UBI FMDV NS-ELISA Kit （United Biomedical Inc. UBI） exhibited a good agreement of 93.1%. The clinical diagnostic potential was evaluated as well.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2005年第5期490-493,共4页
Chinese Journal of Veterinary Science
基金
国家"十五"重大科技专项-食品安全关键技术研究资助项目(2002BA514A-18)
关键词
口蹄疫病毒
非结构蛋白
3B基因
表达
鉴别诊断
酶联免疫吸附试验
FMDV
non-structural protein（NSP）
3B gene
expression
differential diagnosis
enzyme-linked immunosorbent .assay （ELISA）
