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

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.