IL-33纳米抗体的筛选、表达及功能研究

Screening,expression and functional studies of IL-33 nanoantibodies

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。

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.