摘要
为制备抗猪输血传播病毒1型(TTV1)Cap蛋白的单克隆抗体(MAb),本研究采用PCR法扩增TTV1ORF1(1501 nt～2475 nt)编码重组Cap蛋白的C末端截短序列,将PCR产物克隆于pGEX-6P-1载体中,并在大肠杆菌Rosetta(DE3)菌株中诱导表达,获得含GST标签的重组蛋白(rCap),经SDS-PAGE和western blot鉴定表明rCap约为64.0 ku,以包涵体形式存在。用纯化的rCap免疫BALB/c小鼠,采用淋巴细胞杂交瘤技术获得8株稳定分泌抗rCap蛋白的杂交瘤细胞株,MAb亚类均为IgG1/κ型;western blot鉴定表明有7株MAbs与rCap产生特异性反应,另1株MAb呈阴性。为验证8株MAbs与真核表达的Cap蛋白的免疫反应性,将全长与截短的TTV1 ORF1基因片段克隆于pcDNA3.1载体,转染293T细胞进行蛋白瞬时表达,采用免疫过氧化物酶单层细胞染色法检测表明,这8株MAbs均能够与瞬时表达的全长rCap产生特异性反应,其中6株与截短表达的rCap反应。本研究表达的rCap蛋白及制备的MAbs,为该病毒检测方法的建立及抗原表位的鉴定奠定了基础。
Transfusion transmitted virus （TTV） is newly identified virus in humen and animals, to express the capsid （Cap） protein of porcine TTV1 and prepare monoclonal antibody （MAb）, the viral ORF1 gene from 1,501 to 2,475 encoding the truncated Cap protein was amplified by PCR and expressed in E. coli Rosetta （DE3） based on pGEX-6P-1 vector. SDS-PAGE and western blot analysis indicated that the expressed recombinant Cap （rCap） of porcine TTV1 was about 64 ku with GST-tag, mainly in inclusion body form. The SP2/0 cells were fused with the spleen cells from BALB/c mice, which immunized with the purified rCap protein. Eight hybridomas were prepared which stably secreted MAb against the rCap protein. Seven MAbs of which reacted positively with rCap by western blot. For further identification, the MAbs were detected positively by immunoperoxidase monolayer assay in 293T ceUs transfected with recombinant eukaryotic expression plasmid of pcDNA3.1 containing full-length and partial ORF1 gene of TTV1, respectively. The rCap and MAbs provided an useful tool to establish the diagnostic method and epitope analysis for the virus.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2011年第12期974-978,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(30871859)
农业科技成果转化基金(2010GB23260563)
关键词
猪输血传播病毒1型
重组CAP蛋白
单克隆抗体
porcine transfusion transmitted virus type 1
recombinant capsid protein
monoclonal antibody
