摘要
目的克隆人PD-L1胞外区基因并进行原核表达及相应蛋白的抗体制备,为进一步研究PD-1/PD-L1通路及针对该通路进行疾病治疗和药物筛选奠定基础。方法用PCR方法扩增编码人PD-L1胞外区的基因序列,克隆到原核表达载体pET28a(+)中并进行表达,用His抗体为一抗进行Western blotting鉴定并验证结合活性。用纯化的hPD-L1胞外区蛋白作为抗原免疫日本大耳白兔,制备多克隆抗体。通过酶联免疫吸附实验(ELISA)和流式法检测抗体滴度及其特异性。结果原核表达质粒pET28a(+)-hPD-L1成功构建,并可在大肠埃希菌BL21(DE3)中诱导高效表达,得到的hPD-L1胞外区蛋白经SDS-PAGE和Western blotting鉴定正确。用纯化蛋白免疫日本大耳白兔,制备的多克隆抗体具有较强的免疫结合活性,抗体滴度可达105。结论鉴定和纯化了原核表达的hPD-L1蛋白,制备的相应多克隆抗体能够检测真核细胞表达的hPD-L1蛋白,为进一步研究PD-1/PD-L1生物学活性奠定了实验基础。
Objective Programmed death ligand 1 （PD-L1） is an important ligand of programmed death 1 （PD-1）. PD-L1 conjugating to PD-1 confers negative signals to activate lymphocytes,resulting in T/B cell anergy and immune supression. It is very important to know how PD-L1/PD-1 signal pathway works. The present study is focused on PD-L1 protein expression and its activity. Objective To express the extracellular region of PD-L1,purify the protein which acts as an immunogen to generate PD-L1 antibody in rabbit,and verify its activity. Methods The prokaryotic expression plasmid pET28a（＋） /hPD-L1 was constructed by using molecular technique,and then it was transformed to E. coli BL21 （DE3） to induce protein expression with IPTG. The expressed products were identified by SDS-PAGE and Western blot. The purified recombinant protein was used as the antigen to immunize rabbits for 3 times for preparation of polyclonal antibody. The antiserum specificity and titer of hPD-L1 were detected by ELISA and flow cytometry （FCM）. Results The recombinant hPD-L1 extracellular domain protein was expressed and purified. It was a protein with about 30kD molecular weight as analyzed by SDS-PAGE. A high titer antibody （1：105） was obtained by immunizing rabbit with the purified protein. The data of FCM demonstrated that the antiserum had high affinity to PD-L1 expressed on the membrane of PD-L1 transfected cells. Conclusion The purified protein hPD-L1 has been successfully obtained,which possesses a high antigenicity. The prepared polyclonal antibody shows high specificity and titer in immunized rabbits. The preparation of recombinant hPD-L1 and its polyclonal antibody has provided reliable tools for the future study on the biologic activity of PD-1/PD-L1.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第8期997-999,共3页
Medical Journal of Chinese People's Liberation Army
基金
2008年湖北省自然科学基金(ZRY1279)
