Screening And Identification Of Anti-FSH Nanobodies Using Phage Display And High-throughput Sequencing

Follicle-stimulating hormone(FSH)is a kind of gonadotropin hormone secreted by basophilic basophilic cells.The main function is to promote granulocyte hyperplasia,stimulate steroid production,regulate gametocyte development and maturation,Is one of the major hormones in the hypothalamus-pituitary-gonadal axis.The study found that FSH also plays an important role in reproductive system tumors,osteoporosis and tumor angiogenesis,suggesting that FSH may serve as a new target for the treatment and diagnosis of these diseases.FSH consists of ? and ? subunits,molecular weight of about 30 KD,and CG,TSH and LH share ? subunit.The beta subunit contains a 118 amino acid residue that is primarily responsible for the interaction with its receptor follicle stimulating hormone receptor(FSHR).FSH ? subunits of 1 ~ 15 aa,33 ~ 53 aa,51 ~ 65 aa,81 ~ 95 aa and FSHR have a high binding,especially in the FSH33-53 aa binding the strongest,the most widely studied.A number of studies have been used to target binding of tumor cells,and good results have been obtained.Nanobodies are the smallest single domain antibodies derived from the heavy chain variable region of Camelidae.Presently through phage display technology.However,there are some shortcomings in the traditional phage display library,lack of representation of the cloned representation,and difficulty in the screening process.High-throughput technology can be parallel to hundreds of thousands to millions of DNA molecules in sequence,covering almost all of the B cell capacity(107),to solve the traditional phage library screening restrictions.The high and low throughput sequencing techniques were used to sequencing the phage before and after screening,and the antibody gene sequence was obtained by enriching the antibody sequence before and after.In this study,FSH ? 33-53 peptide was used to screen the library,and the specific Nanobody was screened by high-throughput sequencing technology and assisted phage display technology.This study mainly carried out three aspects:1.VHH phage display of natural library construction and affinity screeningTwenty untreated camel blood was collected,total lymphocytes were isolated,total RNA was extracted,and cDNA was obtained by reverse transcription.The VHH gene was obtained by nested PCR and digested with PstI / Not I.It was successfully constructed on pMECS vector and transformed into TG1 cells to construct natural phage camel library.The capacity of the natural library is 1.6 × 109(CFU)/ mL.In this library,the input polyclonal phage was obtained by amplification of VCSM13 helper phage.Through the four-round screening,using monoclonal phage ELISA for identification,selection of positive monoclonal sequencing.The positive RNA antibody sequences VHH-FA7,VHH-FC7 and VHH-FB8 were subcloned into prokaryotic expression vector pET22 b and transformed into BL21(DE3)cells for induction,expression and purification.The binding activity and affinity of Nanobody and FSH?33-53 were measured by ELISA.The results showed that the binding ability of VHH-FA7,VHH-FC7,VHH-FB8 purified protein and FSH?33-53 was not high in monoclonal phage ELISA positive.The binding capacity of VHH-FA7 was about 0.3 when compared with FSH?33-53 at 10 ug / mL,and there was no significant difference compared with negative BSA at the concentration of 1ug / mL.2.High-throughput sequencing was used to identify and screen the phage sequences of the library before and after affinity screeningAfter four rounds of phage library screening,the first round of the input colonies and the fourth round of the phage output were subjected to high throughput sequencing.Five VHG-1296,VHH-6254,VHH-27036,VHH-40740 and VHH-32180 were obtained by biological analysis.Five antibody genes were constructed on pET22 b plasmid and then transformed into BL21(DE3)Cells,IPTG induced crude protein,purified by affinity chromatography on Ni column.FSH ? 33-53 concentration of 10 ug / ml as a primary antibody,secondary antibody for streptomycin-HPR(1: 2500).TMB was measured and the OD value was measured at 450 nm.The binding capacity of the antibody VHH protein tothe antibody was determined according to the OD value.Results: The binding capacity of VHH6254 at 5ug / ml was about 0.4,while that of VHH40740,VHH1296,VHH32180 and VHH40740 was 5 ?g / ml compared with that of FSH? and negative BSA.The binding capacity of antigen FSH?33-53 is low.3.Transfection of FSHR binding peptide into camel nanobody antigen-binding region by affinity transfer method Rapid acquisition of anti-FSHR antibodyThe FSHR binding peptide FSH33-53 coding sequence was transplanted into the CDR1 and CDR3 regions of the Nanobody cAbBCII10,respectively,named VHH-hFSH1 and VHH-hFSH3.And cloned into pET22 b vector,respectively,transformed into E.coli BL21(DE3).IPTG induction and Ni ion affinity chromatography to obtain recombinant single domain antibody protein.The binding and specificity of purified cAbBCII10,VHH-hFSH1,VHH-hFSH3 and FSHR were identified by ELISA.Results: The VHH-hFSH1,VHH-hFSH3 and cAbBCII10 proteins obtained by framework transplantation were soluble in the interstitial cells.ELISA experiments showed that VHH-hFSH3 obtained by grafting FSH33-53 to CDR3 of cAbBCII10 had specific binding to FSHR activity.CONCLUSIONS: cAbBCII10 has the potential to be a non-antibody affinity transfer backbone,but requires further optimization of the graft sequence to achieve high affinity for the antibody.This study shows that the use of high-throughput sequencing technology and bioinformatics analysis can be obtained and prepared with a certain binding force of anti-FSH?33-53 camel nano-antibody,indicating the use of high-throughput sequencing technology may find new nano antibody.However,to obtain high affinity antibodies also need to combine immunization before the antibody abundance analysis,as well as the experimental conditions to optimize and deepen the data mining.