Liu Hui-rong, Zeng Wu-wei, Zhou Bing, Shen Le, Wu Gang, Xue Hong, Chen Bao-sheng
Bioengineering College, Inner Mongolia Agricultural University, Hohhot 010018, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 May;24(5):471-4.
To clone human cholesteryl ester transfer protein (CETP) cDNA, express and purify human CETP in E.coli and prepare CETP-specific rabbit antiserum.
by RT-PCR method, the encoding sequence of human cholesteryl ester transfer protein (CETP) was cloned into pET-30b(+) vector. Then BL21 (DE3) of E.coli transformed with recombinant vector pET-CETP was induced to express CETP in high level by IPTG. The expressed protein was purified from SDS-polyacrylamide gel, and the antiserum against CETP was raised in rabbit. The titer and specificity of rabbit antiserum were evaluated by ELISA, Western blot and immunofluorescence assay.
The results of SDS-PAGE showed that CETP was expressed in the form of inclusion bodies in BL21(DE3) and the best expression time was about 4 hours. The titer of the rabbit antiserum prepared with CETP purified from SDS-PAGE was 1:5. 12 x 10(5) and the antiserum reacted specifically with CETP expressed in BL21 (DE3) and COS7 cells.
The preparation of the specific rabbit antiserum against CETP will be valuable for the study on the structure and function of human CETP.
克隆人胆固醇酯转运蛋白（CETP）cDNA，在大肠杆菌中表达并纯化人CETP，制备抗CETP的兔抗血清。
采用RT-PCR法，将人胆固醇酯转运蛋白（CETP）编码序列克隆至pET-30b(+)载体。用重组载体pET-CETP转化大肠杆菌BL21(DE3)，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导高水平表达CETP。从十二烷基硫酸钠-聚丙烯酰胺凝胶中纯化表达的蛋白，并在兔体内制备抗CETP血清。通过酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法（Western blot）和免疫荧光分析评估兔抗血清的效价和特异性。
十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示，CETP在BL21(DE3)中以包涵体形式表达，最佳表达时间约为4小时。用从SDS-PAGE纯化的CETP制备的兔抗血清效价为1:5.12×10⁵，该抗血清与在BL21(DE3)和COS7细胞中表达的CETP特异性反应。
制备抗CETP的特异性兔抗血清对研究人CETP的结构和功能具有重要价值。
