[Preparation and identification of anti-follicle-stimulating hormone receptor nanobodies].

OBJECTIVE

To prepare camel derived nanobodies which specifically bind to follicle-stimulating hormone receptor (FSHR).

METHODS

The FSHR gene fragment (fshr234) was expressed in E.coli as antigen for affinity screening against VHH phage display library constructed from Xinjiang Bactrian camel. After confirmed by DNA sequencing, the vhh gene fragments of interest were subcloned into pET30a expression vector, and then were used to transform E.coli BL21(DE3). After IPTG induction, 6×His and c-Myc tagged fusion nanobodies were expressed. The nanobodies were purified by Ni-ion affinity chromatography. The binding specificity of nanobodies with His-FSHR234 was determined by ELISA.

RESULTS

By enrichment screening with the antigen His-FSHR234, the 28 clones showed VHH sequence identities in DNA sequencing from 40 randomly selected binding clones. The 4 clones were subcloned into pET30a vector and confirmed as expected size of inserts by PCR and endonuclease digestion. The 4 expressed and affinity purified recombinant nanobodies namely VHHFSHR;-06, VHHFSHR;-25, VHHFSHR;-30 and VHHFSHR;-50 showed single band at Mr; 31 000, 26 000, 25 000 and 26 000 on SDS-PAGE, respectively. ELISA results showed that 4 nanobodies could bind to FSHR234 specifically, in which VHHFSHR;-06 showed the highest antigen binding activity.

CONCLUSION

By screening camel VHH phage display library with His-FSHR234 antigen, one nanobody, VHHFSHR;-06 with relatively high antigen binding activity has been produced and identified.