摘要
目的:以白细胞介素15(IL-15)为靶点,研制类风湿性关节炎免疫治疗蛋白疫苗。方法:将N端融合破伤风类毒素(TT)表位的人IL-15基因克隆至带有His标签的原核表达载体pQE-30上,转化大肠杆菌M15,经IPTG诱导表达,获得重组人TT-IL-15融合蛋白(简称rtIL-15);目的蛋白经镍柱亲和层析纯化后,用Western印迹和HPLC进行鉴定;将rtIL-15与氢氧化铝佐剂混合,免疫BALB/c小鼠,检测疫苗的免疫原性。结果:双酶切鉴定和核苷酸序列测定结果表明重组融合表达质粒pQE-30-TT-IL-15构建正确,重组蛋白的表达量达到菌体总蛋白的20%,主要以包涵体形式表达,经过纯化、复性后,目的蛋白纯度达到95%以上;蛋白疫苗免疫小鼠后,能诱导高滴度的特异性的IL-15抗体。结论:在大肠杆菌中表达了重组人IL-15融合蛋白疫苗,并具有良好的免疫原性。
Objective： To express human interleukin-15（IL-15） in Escherichia coli and determine the immunogenicity of recombinant IL-15 fusion protein. Methods： IL-15 gcne, fused with tetanus toxoid（TT） epitope in N terminal, was cloned into prokaryotic expression vector pQE-30 with His fusion labeling, and the constructed plasmid was transformed to E.coli M15 for expression under induction of IPTG. The target protein was purified by affinity chromatography and identified by Western blot and IPTG. The protein vaccine was immunized to BALB/c mice to identify the immunogenicity. Results： DNA nucleotide sequencing and restriction analysis proved that the recombinant plasmid pQE-30-TT-IL-15 was constructed correctly. The target protein was up to 20% of total induced protein and mainly existed in inclusion body. The purify of the target protein was up to 95% after purification and renaturation. High titer antisera against IL-15 was obstained by immunizing BALB/c mice with the purified Tr-IL- 15 protein vaccine. Conclusion： The recombinant TT-IL-15 fusion protein was successfully expressed in E.coli and induced humoral immune response in mice.
出处
《生物技术通讯》
CAS
2014年第3期349-352,共4页
Letters in Biotechnology
基金
国家自然科学基金(81001182)
