Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Department of Biochemistry and Molecular Biology, Medical School of Southeast University, Nanjing, 210009, China.
Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Department of Biochemistry and Molecular Biology, Medical School of Southeast University, Nanjing, 210009, China.
Mol Immunol. 2019 May;109:12-19. doi: 10.1016/j.molimm.2019.02.022. Epub 2019 Mar 5.
The persistent infection of high-risk human papillomavirus (HPV) is one of the most common causes of cervical cancer. It is well documented that expression of two oncogenes (E6/E7) plays a key role in tumor progression. HPV16E7 -targeting via nanobody (Nb) therefore could be beneficial for HPV16-associated cancer diagnosis and therapy. In this work, phage-display approach was employed to select the high affinity HPV16E7-specific Nb. Firstly; a high-quality immune library was constructed. After three round of biopanning, high-affinity HPV16 E7-specific nanobodies were retrieved. By phage ELISA and sequencing, four different sequences of anti- HPV16E7 nanobodies were selected. Then recombinant nanobody Nb2 was cloned and expressed in E. coli, and the specificity and thermal stability of purified Nb2 was evaluated. To examine the potential of Nb2 as an inhibitor of E7 function, Nb2 was expressed within HPV16 positive cells. Proliferation assay showed that the intracellular expressed Nb2 as an intrabody can decrease the growth of HPV16-positive cells. The results indicate that Nb2 as an intracellular antibody directed towards HPV oncoprotein E7 has great promise in applications for the therapy of HPV16-associated disease.
高危型人乳头瘤病毒（HPV）的持续感染是宫颈癌最常见的病因之一。有充分的文献记载表明，两种致癌基因（E6/E7）的表达在肿瘤进展中起着关键作用。因此，针对 HPV16E7 的纳米抗体（Nb）靶向治疗可能有益于 HPV16 相关癌症的诊断和治疗。在这项工作中，我们采用噬菌体展示技术来筛选高亲和力的 HPV16E7 特异性纳米抗体。首先，构建了一个高质量的免疫文库。经过三轮生物淘选，我们获得了高亲和力的 HPV16E7 特异性纳米抗体。通过噬菌体 ELISA 和测序，我们选择了四个不同序列的抗 HPV16E7 纳米抗体。然后，我们将重组纳米抗体 Nb2 克隆并在大肠杆菌中表达，并评估了纯化的 Nb2 的特异性和热稳定性。为了研究 Nb2 作为 E7 功能抑制剂的潜力，我们在 HPV16 阳性细胞中表达了 Nb2。增殖实验表明，细胞内表达的 Nb2 作为一种内抗体可以抑制 HPV16 阳性细胞的生长。结果表明，Nb2 作为一种针对 HPV 致癌蛋白 E7 的细胞内抗体，在治疗 HPV16 相关疾病方面具有很大的应用前景。
