卵泡刺激素受体FSHR234纳米抗体的分离纯化及多克隆抗体的制备

Isolation and identification of follicle-stimulating hormone receptor (FSHR234) nanobody and polyclonal antibody

摘要:

【背景】卵泡刺激素受体(Follicle-stimulating hormone receptor，FSHR)在人体成熟破骨细胞及单核细胞表面表达，成为阻断卵泡刺激素(FSH)作用的潜在靶点，制备亲和力较高的FSHR抗体已成为靶向治疗FSH相关疾病的切入点。【目的】构建FSHR重组载体获得表达量较高的可溶性蛋白，进一步制备骆驼多克隆抗体及其纳米抗体。【方法】利用XhoⅠ和BamHⅠ双酶切重组质粒pET30a-FSHR234，将目的基因fshr构建于pGEX-4T-1载体并进行原核表达，利用Western blot及ELISA检测目的蛋白的配体结合能力。免疫新疆双峰驼制备骆驼多克隆抗体，并利用噬菌体展示技术筛选获得FSHR纳米抗体。细胞免疫化学法检测FSHR抗体在coav-3的表达情况。【结果】构建了pGEX-4T-1-FSHR234重组质粒，获得了表达量较高的FSHR234可溶性蛋白；并构建了5株FSHR纳米抗体，鉴定出一株结合能力较强的纳米抗体，命名为VHH-3F9。【结论】制备的骆驼FSHR多克隆抗体效价达1:128 000，筛选获得的VHH-3F9纳米抗体能与FSHR234具有较高的结合性，且两者均能表达于coav-3细胞表面。

Abstract:

[Background] follicle-stimulating hormone receptor (FSHR) is secreted by the anterior pituitary cells which expressed in the cell surface of human mature osteoclasts and mononuclear, was regarded as potential targets for blocking FSH. [Objective] For the purpose of obtaining soluble FSHR protein in order to acquire FSHR nanobody with high affinity for the treatment of related diseases caused by FSH and bone loss. [Methods] Recombinant plasmid pET30a-FSHR234 was double enzyme cut by XhoⅠand BamHⅠ, fshr gene was constructed to the pGEX-4T-1 vector and expressed in the prokaryotic cell. Its’ binding capacity was determined by Western blot and ELISA. Immunized Xinjiang camels were used to prepare polyclonal antibody. Phage display technology was used to obtain FSHR nanobodies of high affinity. The expression of coav-3 was detected by cellular immunochemistry. [Results] In this study, recombinant pGEX-4T-1-FSHR was constructed successfully, and the soluble FSHR234 protein expressed with high concentration. Five FSHR nanobodies were obtained successfully by mean of subclone. [Conclusion] The titer of camel FSHR polyclonal antibody prepared in the experiment was 1:128 000. VHH-3F9 nanobody could combine FSHR234 at a low concentration and its binding capacity enhanced with increasing FSHR234 concentration. In addition, both FSHR polyclonal antibody and VHH-3F9 nanobody could expressed on the surface of coav-3.