cDNA cloning and prokaryotic expression of human cholesteryl ester transfer protein and preparation of its antiserum

• Published in: Xi bao yu fen zi mian yi xue za zhi Vol. 24; no. 5; p. 471
• Main Authors: Liu, Hui-rong, Zeng, Wu-wei, Zhou, Bing, Shen, Le, Wu, Gang, Xue, Hong, Chen, Bao-sheng
• Format: Journal Article
• Language: Chinese
• Published: China 01.05.2008
• Subjects: Animals; Antibodies, Monoclonal; Blotting, Western; Capsid Proteins - immunology; Cholesterol Ester Transfer Proteins - genetics; Cholesterol Ester Transfer Proteins - immunology; Cloning, Molecular; DNA, Complementary - analysis; DNA, Complementary - genetics; DNA, Complementary - immunology; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Gene Expression - drug effects; Genetic Vectors; Humans; Isopropyl Thiogalactoside - pharmacology; Rabbits; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA
• ISSN: 1007-8738

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 wil