Xia Xueqin, M Muyassar, Zhai Tiantian, Li Jiangwei
College of Life Science and Technology, Xinjiang University, Urumqi, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Aug;29(8):829-33.
To prepare camel derived nanobodies which specifically bind to follicle-stimulating hormone receptor (FSHR).
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.
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.
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.
制备特异性结合促卵泡激素受体（FSHR）的骆驼源纳米抗体。
将FSHR基因片段（fshr234）在大肠杆菌中表达作为抗原，用于筛选从新疆双峰驼构建的VHH噬菌体展示文库。经DNA测序确认后，将感兴趣的vhh基因片段亚克隆到pET30a表达载体中，然后用于转化大肠杆菌BL21(DE3)。经IPTG诱导，表达带有6×His和c-Myc标签的融合纳米抗体。通过镍离子亲和层析纯化纳米抗体。采用ELISA法测定纳米抗体与His-FSHR234的结合特异性。
用抗原His-FSHR234进行富集筛选，从40个随机选择的结合克隆中，28个克隆在DNA测序中显示VHH序列一致性。将4个克隆亚克隆到pET30a载体中，通过PCR和内切酶消化确认插入片段大小符合预期。4个表达并经亲和纯化的重组纳米抗体，即VHHFSHR;-06、VHHFSHR;-25、VHHFSHR;-30和VHHFSHR;-50，在SDS-PAGE上分别显示Mr为31 000、26 000、25 000和26 000的单一条带。ELISA结果显示，4种纳米抗体均可特异性结合FSHR234，其中VHHFSHR;-06的抗原结合活性最高。
用His-FSHR234抗原筛选骆驼VHH噬菌体展示文库，制备并鉴定出一种具有较高抗原结合活性的纳米抗体VHHFSHR;-06。
