【摘要】 目的:白细胞介素33（interleukin 33,IL-33）/IL-1受体相关的致瘤性受体2（suppression of tumorigenicity 2, ST2）信号通路在肿瘤的发展中起着重要作用，IL-33升高会促进肿瘤细胞的增殖。本研究旨在筛选和表达靶向IL-33的纳米抗体，通过与IL-33结合阻断IL-33/ST2信号通路的转导，调节炎症反应并防止肿瘤恶化。方法:通过淘洗、富集验证和筛选从大容量天然噬菌体纳米抗体文库中筛选与IL-33特异性结合的纳米抗体序列，获得2个氨基酸序列不同的IL-33纳米抗体(Nb_IL-33);利用PCR技术将Nb_IL-33纳米抗体序列从噬菌体质粒pMECS中扩增出来，通过构建质粒将纳米抗体序列连接到表达载体pMal-c4x中并转化至表达菌株BL21（DE3）,获得Nb_IL-33与MBP标签蛋白可溶性融合表达菌株；使用Ni-NTA亲和层析的方法纯化Nb_A1和Nb_E12融合蛋白；使用ELISA法检测Nb_IL-33的特异性、亲和力和热稳定性；使用CCK8法检测Nb_IL-33对IL-33诱导人乳腺癌细胞增殖的抑制作用。结果:从天然噬菌体纳米抗体文库中筛选与IL-33特异性结合的纳米抗体序列，获得2个氨基酸序列不同的Nb_IL-33纳米抗体，分别命名为Nb_A1和Nb_E12;并在大肠杆菌（E.coli）BL21中进行表达，通过Ni-NTA亲和层析获得纯度90%以上的2种纳米抗体。ELISA检测表明，Nb_A1和Nb_E12均可以与IL-33结合，其亲和力常数Ka值分别为（6.068±2.58）×10⁵ mol/L和（2.17±0.37）×10⁶ mol/L。CCK8检测证明了Nb_A1和Nb_E12对IL-33诱导人的乳腺癌细胞增殖均有抑制作用，且Nb_A1的抑制作用大于Nb_E12。结论:本研究从天然噬菌体纳米抗体文库中筛选获得了2个靶向IL33抗原的纳米抗体序列，并构建了Nb_IL-33融合蛋白表达菌株；通过Ni-NTA亲和层析纯化得到纯度大于90%的非融合蛋白Nb_A1和Nb_E12,且对其生物学功能进行初步研究，为靶向IL-33的肿瘤治疗策略提供实验数据。更多还原

【Abstract】 Objective:The interleukin-33（IL-33）/suppression of tumorigenicity 2（ST2） signaling pathway plays an important role in the development of tumors. Increased IL-33 levels promote the proliferation of tumor cells. This article aims to screen and express nanoantibodies targeting interleukin IL-33, block the transduction of the IL-33/ST2 signaling pathway by binding to IL-33, regulate inflammatory responses, and prevent tumor deterioration. Methods: Nanoantibody sequences that specifically bind to interleukin IL-33 were screened from a large-capacity natural phage nanoantibody library by panning, enrichment verification and screening, and two Nb_IL-33 nanoantibodies with different amino acid sequences were obtained; the Nb_IL-33 nanoantibody sequence was amplified from the phage plasmid pMECS by PCR technology, and the nanoantibody sequence was connected to the expression vector pMal-c4x by plasmid construction method and transformed into the expression strain BL21（DE3） to obtain the soluble fusion expression strain of Nb_IL-33 and MBP tag protein: Ni-NTA affinity chromatography was used to purify Nb_A1 fusion protein and Nb_E12 fusion protein; ELISA method was used to detect the specificity, affinity and thermal stability of Nb_IL-33; CCK8 method was used to detect the inhibitory effect of Nb_IL-33 on IL-33-induced human breast cancer cell proliferation. Results: By screening the nanoantibody sequence that specifically binds to interleukin IL-33 from the natural phage nanoantibody library, two Nb_IL-33 nanoantibodies with different amino acid sequences were obtained, named Nb_A1 and Nb_E12 respectively; they were expressed in E.coli BL21, and two nanoantibodies with a purity of more than 90% were obtained by Ni-NTA affinity chromatography. ELISA showed that both Nb_A1 and Nb_E12 could bind to IL-33, and their affinity constants Ka values were（6.068±2.58）×10⁵ mol/L and（2.17±0.37）×10⁶ mol/L, respectively; CCK8 assay proved that both NbA1 and NbE12 had an inhibitory effect on IL-33-induced human breast cancer cell proliferation, and the inhibitory effect of Nb_A1 was greater than that of Nb_E12. Conclusion:In this study, two IL33-targeting nanoantibody sequences were screened from the natural phage nanoantibody library, and the Nb_IL-33 fusion protein expression strain was constructed. Non-fusion proteins Nb_A1 and Nb_E12 with a purity greater than 90% were purified by Ni-NTA affinity chromatography, and their biological functions were preliminarily studied, laying the foundation for the tumor treatment strategy targeting IL-33.更多还原