人磷脂转移蛋白的原核表达及其抗体的制备与鉴定

Preparation and characterization of the antibody against expressed human phospholipid transfer protein

目的利用大肠杆菌表达人磷脂转移蛋白(PLTP),制备兔抗人PLTP的多克隆抗血清。方法采用RT-PCR技术,将人PLTP蛋白编码序列克隆入pET-30b(+)原核表达载体,转化大肠杆菌,IPTG诱导表达,切胶纯化蛋白后免疫家兔,制备PLTP抗血清。用间接ELISA法、Western blot、细胞免疫荧光检测对抗血清效价及特异性进行鉴定。结果SDS-PAGE分析表明,PLTP基因在大肠杆菌BL21(DE3)菌株的包涵体中高效表达,最佳诱导表达时间为3h;将纯化的蛋白免疫家兔,ELISA法测定的抗血清效价为1∶256000;Western blot及细胞免疫荧光检测显示,抗血清可以与PLTP原核及真核表达蛋白特异结合。结论成功地将PLTP进行了原核表达,制备了高效价、高特异性的兔抗人PLTP抗血清,为PLTP的临床检测及其结构与功能的进一步研究提供了有力工具。

Objective To prepare rabbit antibody against human phospholipid transfer protein （PLTP） using protein expressed in E. coll. Methods By RT-PCR method, the encoding sequence of human PLTP was cloned into pET-30b（ ＋ ） vector. Then BL21 （DE3） of E. eoli transformed with recombinant vector pET-PLTP was induced to express PLTP in high level by IPTG. The expressed protein was purified from SDS-polyacrylamide gel, and the antibody against PLTP was raised in rabbit. The titer and specificity of rabbit antibody were evaluated by ELISA, Western blotting, and immunofluorescence assay. Results The results of SDS-PAGE showed that PLTP was expressed in the form of inclusion bodies in BL21 （DE3） and the best expression time was about 3 hours. The titer of the rabbit antibody prepared with PLTP purified from SDS-PAGE was 1：256 000 and the antibody could reacted specifically with PLTP expressed in BL21 （DE3） and COS7 cells. Conclusion The preparation of the specific rabbit antibody against PLTP will benefit the clinical detection of PLTP, as well as the study on the structure and function of human PLTP.