Ma Jifu, Bodai Bakhet, Ma Zhongmei, Khalembek Kezerbek, Xie Jingang, Kadyken Rizabek, Baibatshanov Mukhtar, Kazkhan Oralhazi
College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, 832000, China.
Agricultural Development Service Center of Kalabulegen Township, Fuyun County, Altay Region, Xinjiang, 836103, China.
Heliyon. 2024 Aug 12;10(17):e36180. doi: 10.1016/j.heliyon.2024.e36180. eCollection 2024 Sep 15.
Inhibin is a member of the transforming growth factor family that influences reproduction in animals.
The purpose of this study was to obtain nanobodies from the phage antibody library constructed by us that can specifically bind to inhibin α-subunit.
In this study, camels were immunized with Kazakh sheep inhibin-α protein that expressed in BL21 , and the camel VHH nanobody phage display library was prepared using nested PCR. The nanobodies specifically binding to inhibin α-subunit in the library were screened by three rounds of immunoaffinity screening and phage enzyme-linked immunosorbent assay (phage ELISA). The functions of the selected nanobodies were identified using molecular simulation docking, ELISA affinity test, and sheep immunity test.
A nanobody display library was successfully constructed with a capacity of 1.05 × 10 CFU, and four inhibin-α-subunit-specific nanobodies with an overall similarity of 69.34 % were screened from the library, namely, Nb-4, Nb-15, Nb-26, and Nb-57. The results of molecular simulation docking revealed that four types of nanobodies were complexed with inhibin-α protein mainly through hydrophobic bonds. Immunity tests revealed that the nanobody Nb-4 could effectively inhibit sheep inhibin A/B and could significantly improve the FSH level in sheep.
Four inhibin α-subunit-specific nanobodies with biological functions were successfully screened. To the best of our knowledge, this is a new reproductive immunomodulatory pathway of inhibin α-subunit, which may change the secretion of FSH in the ovary, thus changing the estrous cycle of organisms.
抑制素是转化生长因子家族的成员，影响动物的生殖。
本研究旨在从我们构建的噬菌体抗体库中获得能特异性结合抑制素α亚基的纳米抗体。
本研究用在BL21中表达的哈萨克绵羊抑制素α蛋白免疫骆驼，采用巢式PCR制备骆驼VHH纳米抗体噬菌体展示库。通过三轮免疫亲和筛选和噬菌体酶联免疫吸附测定（噬菌体ELISA）从库中筛选出特异性结合抑制素α亚基的纳米抗体。利用分子模拟对接、ELISA亲和力测试和绵羊免疫试验鉴定所选纳米抗体的功能。
成功构建了库容为1.05×10CFU的纳米抗体展示库，从库中筛选出4种抑制素α亚基特异性纳米抗体，总体相似度为69.34%，即Nb-4、Nb-15、Nb-26和Nb-57。分子模拟对接结果显示，4种纳米抗体与抑制素α蛋白主要通过疏水键结合。免疫试验表明，纳米抗体Nb-4能有效抑制绵羊抑制素A/B，并能显著提高绵羊体内促卵泡素水平。
成功筛选出4种具有生物学功能的抑制素α亚基特异性纳米抗体。据我们所知，这是抑制素α亚基一种新的生殖免疫调节途径，可能改变卵巢中促卵泡素的分泌，从而改变生物体的发情周期。
