前列腺特异性膜抗原(PSMA)纳米抗体基因的原核表达及天然噬菌体纳米抗体库筛选

Prokaryotic expression of prostate-specific membrane antigen(PSMA)nanoantibody gene and screening of natural phage nanoantibody library

目的筛选出抗前列腺特异性膜抗原(PSMA)的纳米抗体。方法利用天然噬菌体纳米抗体库,以PSMA为靶抗原进行三轮的淘选,采用ELISA鉴定阳性克隆,并进行测序分析,将筛选到的阳性克隆基因序列插入pET28a原核表达载体中,并转化至大肠杆菌BL21中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白的表达,利用Ni柱进行亲和纯化,通过SDS-PAGE验证纯化产物。结果筛选获得4条PSMA纳米抗体序列重链可变区1(VHH1)、VHH2、VHH3、VHH4,VHH1序列未能获得明确的原核表达蛋白,VHH2、VHH3、VHH4蛋白均表达。纯化后获得了纯度高、相对分子质量(Mr)约为17000的抗PSMA纳米抗体。结论通过天然噬菌体纳米抗体库淘筛,成功获得抗PSMA的纳米抗体序列并进行了原核表达和纯化。

Objective To screen nanobodies against prostate specific membrane antigen(PSMA). Methods Based on the naive phage display library, three rounds of screening were performed targeting the PSMA antigen, and positive clones were identified by ELISA and sequencing was performed. The positive cloned gene sequence was inserted into the pET28a prokaryotic expression vector and transformed into E.coli BL21. The expression of the recombinant protein was induced by IPTG and purified using Ni column, with the purified product verified by SDS-PAGE. Results Four PSMA nanobodies VHH1, VHH2, VHH3 and VHH4 were obtained by screening. The VHH1 failed to obtain protein expression, while the VHH2, VHH3 and VHH4 proteins were expressed. The purity of anti-PSMA nanobodies showed high and relative molecular mass(Mr) of about 17 000. Conclusion The sequence of anti-PSMA nanobody was successfully obtained by screening the naive phage nanobody library and were subjected to prokaryotic expression and purified.