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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 clone, express and purify human non-structural protein NS1 of Bocavirus (HBoV) and prepare anti-NS1 polyclonal antibody. Methods: NS1 gene of HBoV nonstructural protein was amplified by PCR and cloned into pMAL-c2X expression vector. The recombinant plasmid was transformed into E. coli DH10B and induced by IPTG. The expressed fusion protein was purified by Amylose Resin affinity chromatography and immunized New Zealand rabbits to prepare polyclonal antibodies. Antibody titers were measured by indirect ELISA. Results: The fusion protein MBP-NS1 was prokaryotic expressed and its polyclonal antibody was obtained. The titer of the antibody reached 1:32000. CONCLUSION: The fusion protein was expressed and purified in prokaryotic expression system to prepare anti-NS1 polyclonal antibody, which provided a reliable tool for further study on the transcription and translation mechanism of the non-structural protein gene of the virus.