Identification Of Follicle-stimulating Hormone Receptor Specific Nanobodies Though Next-generation Sequencing

Follicle stimulating hormone receptor(Follicle-stimulating hormone receptor,FSHR) not only play an important role in the reproductive system, It also found specifically expressed in a variety of tumor vascular endothelial cell which is related with tumor angiogenesis.Besides,FSHR may play an important role in the acceleration of bone loss in postmenopausal women. So FSHR is a potential target in the diagnosis and treatment of cancer and osteoporosis. Nanobodies are the variable domain alone of heavy chain antibodies with approximate molecular weight of 15 kDa,It has many advantages over conventional antibodies,Such as it's weekly immunogenic and high stability.The main method of preparing nanobody is phage display library technology at present,but it also time-consuming,screening results uncertainty and difficult to get high affinity antibodies.High-throughput sequencing technology,also called "Next-generation" sequencing technology, the hundreds of thousands to one million DNA molecule were sequenced in a parallel by NGS.Antigen-specific antibody sequences can be picked from immunization antibody library after High-throughput sequence analysis.The main purpose of this study is to prepare anti-FSHR camelid nanobodies by high-throughput sequencing technology and verify their specificity and affinity,Discussion the value of discovery and identification of antibodies By high-throughput sequencing technology.There are two main part research contents in the thesis:1.High-throughput DNA sequencing and analysis of camel lymphocytes heavy chain variable region gene bank after FSHR immunizationFirstly,FSHR recombinant protein was expressed in E.coil BL21 by IPTG induction. Used the Purified recombinant protein to periodically immune the Xinjiang camel. Collect camel blood sample before and after the sixth immunization to Evaluate Anti-FSHR antibody titer by Enzyme-Linked Immunosorbant Assay. Whenthe antibody titer reaches the requirement,collect 300 mL of blood from camel jugular vein, peripheral blood lymphocytes isolated from camel blood. Meanwhile,Total RNA was extracted form peripheral blood lymphocytes,The VHH gene fragments were PCR amplified with cDNA as a template.Finally, The desired antibody VHH sequence by bioinformatics software generation sequencing data for depth analysis.Xinjiang Bactrian camel serum anti-FSHR antibody titers reach 1: 128,000 after the sixth immunization with FSHR. High-throughput sequencing provided 2,344,304 reads after trimming and merge,839,219 in-frame VHH sequences were deduced from the deep sequencing data. Only runs in which 80% of sequences have a Phred score >30% were used. CDR3 length determination and Frequencies were analysised,the top tenth of most Frequently CDR3 sequences was picked to nanobody expression.2.Identification and generation of anti-FSHR nanobodiesThe expected VHH coding sequences obtained from NGS were cloned into the pET28 b expression vector,named BL21-pET28b-S1-10. Recombinant nanobodies were expressed in BL21(DE3) strain by 0.2 mM IPTG induction and purified with Ni-affinity chromatography. The Binding ability and affinity of expressed nanobodies were identified by anti-c-myc mAb in ELISA assay and Western Blot for detection of their binding with FSHR antigen.Besides,Binding ability of S6 and S9 antibodies to eukaryotic cells Caov-3 which express FSHR was detected by immunocytochemistry.The top tenth VHH gene fragments was cloned into pET28 b vector and induced with 0.2 mM IPTG and 30 ?. S5?S6?S7 antibody proteins were recovered as inclusion bodies, S2,S3,S8,S9 and S10 antibody were secretary expression.At the same time,S1 and S4 antibody proteins were not expression in Ecoli BL21.The approximate molecular weight of those VHH proteins are 19 kDa.The Enzyme-linked immunosorbent assay and Western blot reveled that S6 and S9 antibody specific binding to FSHRantigen.However,immunocytochemistry indicated that the S6 and S9 antibodies have no binding to eukaryotic cells Caov-3.In Conclusion, FSHR can stimulate higher humoral immune response in the Xinjiang Bactrian camels body. The anti-FSHR nanobodies With a certain binding ability were obtained after the high-throughput sequencing combination with bioinformatics analysis,It has the potential to discover new nanobodies used the high-throughput sequencing technology. But in order to obtain high-affinity antibody,The abundance of pre-immune antibody sequences were also needed to analysis, Besides,it's important to optimize the experimental conditions and dig high-throughput sequencing data deeper.