摘要
目的获得抗卵泡刺激素受体(FSHR)的纳米抗体。方法使用原核表达的重组蛋白His-FSHR234对新疆双峰驼单域抗体噬菌体展示文库进行亲和筛选,将筛选获得的重链抗体的可变区(vhh)基因亚克隆至pET30a表达载体,转化E.coli BL21(DE3),IPTG诱导表达VHH重组蛋白,镍离子亲和层析柱纯化获得纳米抗体。ELISA检测纳米抗体的抗原结合活性。结果 His-FSHR234筛选富集后,随机挑选40个克隆进行鉴定,其中28个为阳性克隆,挑选4个vhh基因进行克隆,PCR和酶切鉴定目的基因大小与预计相符。SDS-PAGE显示,VHH FSHR-06、VHH FSHR-25、VHH FSHR-30、VHH FSHR-50分别在相对分子质量(M r)31 000、26 000、25 000、26 000有目的条带。ELISA检测显示,4个纳米抗体对His-FSHR234重组蛋白均具有结合活性,其中VHH FSHR-06的抗原结合活性最高。结论从新疆双峰驼单域抗体噬菌体展示文库中筛选获得了1个具有较高抗原结合活性的抗FSHR的纳米抗体。
Objective To prepare camel derived nanobodies which specifically bind to follicle-stimulating hormone recep-tor （FSHR）. Methods The FSHR gene fragment （fsht234） 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 frag-ments of interest were subcloned into pET30a expression vector, and then were used to transform E. coli BI_21 （DE3）. After IPTG induction, 6 x 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 random-ly 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, VHHFsHRO25, 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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第8期829-833,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
新疆自治区高技术研究发展项目(201110102)
国家自然科学基金(81260333)
