摘要
目的:克隆、表达、纯化人类博卡病毒(HBoV)非结构蛋白NS1,制备抗NS1多克隆抗体。方法:利用PCR扩增HBoV非结构蛋白NS1基因,将其克隆至pMAL-c2X表达载体上,重组质粒转化大肠杆菌DH10B,IPTG诱导表达。表达的融合蛋白经Amylose Resin亲和层析柱纯化后,免疫新西兰大白兔制备多克隆抗体。用间接ELISA法检测抗体效价。结果:原核表达融合蛋白MBP-NS1,并获得了其多克隆抗体,抗体效价达到1∶32000。结论:在原核表达系统中表达、纯化了融合蛋白,制备抗NS1多克隆抗体,为进一步研究该病毒非结构蛋白基因的转录和翻译机制提供可靠的工具。
Objective： To make polyclonal antibody by cloning and expressing the nonstructural protein gene NS1 of the Human bocavirus （HBoV） in Escherichia coli DH10B. Methods： NS1 gene of the HBoV was amplified by PCR and inserted into prokaryotie expression vector pMAL-c2X which contained the maltose binding protein （MBP）, and then it was expressed in Escherichia coli DH10B strain. The target fusion protein was purified by Amylose affinity chromatography, and was used to immunize the New Zealand white rabbit for preparing the corresponding antibody. The titer of the produced antibody was measured by ELISA. Results： The NS1 gene was cloned and expressed in E.coli successfully. The polyclonal antibody was obtained after immunization of the New Zealand rabbit with the purified protein and its titer reached up to 1：32000. Conelusiun： The MBP -NS1 fusion protein was successfully expressed and purified, and the polyclonal antibody was produced, which provides a reliable tool for further study of transcription and translation mechanisms of this gene.
出处
《现代生物医学进展》
CAS
2010年第6期1013-1016,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金(Nos.30670081)
